translation reinitiation and ribosome recycling - phd thesis
TRANSCRIPT
Reinitiation of translation and ribosome recycling are two distinct mechanisms in
Saccharomyces cerevisiae
A dissertation submitted to the
University of Manchester Institute of Science and Technology
(UMIST) for the degree of Doctor of Philosophy
2003
LUKAS RAJKOWITSCH Department of Biomolecular Sciences
Declaration
No portion of this work referred to in this thesis has been submitted in support of an
application for another degree or qualification of this or any other university, or other
institution of learning.
Part of this work has been submitted in July 2003 to the Journal of Molecular Biology
for publication.
All work described in this thesis was carried out in accordance with Control of
Substances Hazardous to Health regulations (COSHH) in their current versions.
2
Dedication
for Heidi, my parents,
and all of you who supported me
on this way forth and back
1% inspiration, 99% transpiration
3
Acknowledgements
ACKNOWLEDGEMENTS I would like to thank Prof John McCarthy for providing me with the opportunity to
perform the work for this study in his laboratory and for the invaluable guidance
throughout its course. I am also grateful to all the other members of the
Posttranscriptional Control Group at UMIST who dramatically reduced the
theoretical and practical obstacles especially at the outset of this work and –
equally important – provided a lively surrounding I enjoyed working in. Special
thanks go to Cristina Vilela for general assistance and guidance during this
project and to Karine Berthelot as well as Carmen Ramirez Velasco for
introducing me to the wonderful worlds of in vitro transcription and mRNA
stability measurements, respectively, among many other aspects. Agoritsa
Varaklioti kindly provided access to computer resources and storage solutions,
and Mara Nardelli along with Sabina Piccinnini shared extraordinary knowledge
of sucrose gradients. Marina Ptushkina did not leave me alone with (RT-)PCR
or other nucleic acid problems, and Tobias von der Haar was always there for
information about translation in general and protein methods in particular.
Finally, John Hughes was at all times remarkably inventive in the fields of
financial administration.
Special thanks to Yoav Arava of Stanford University for providing sucrose
gradient protocols.
4
Abstract
ABSTRACT Historically, translation termination was viewed simply as the last step in the
process of cellular protein synthesis. Recent findings have changed this
perception dramatically, indicating that events at this stage decide about the
fate of the posttermination ribosome and about the degradation of the translated
mRNA. In particular, it remains to be elucidated how terminating ribosomes are
recycled back to start another round of translation. The circularisation model of
the polysomal mRNA suggests that this ribosome recycling might be facilitated
by 5' – 3' interactions mediated by the cap-binding complex eIF4F and the
poly(A) binding protein, Pab1p. In contrast, downstream of a short upstream
open reading frame (uORF) in the 5' untranslated region of a gene,
posttermination ribosomes can maintain the competence to (re)initiate
translation. This study shows that recycling and reinitiation are distinct
processes in Saccharomyces cerevisiae. Recycling via the 3'-UTR was
assessed by restricting ribosome movement along the mRNA using a poly(G)
stretch or the mammalian iron regulatory protein (IRP1) bound to the iron
responsive element (IRE). Although 3'-UTR structures were found to influence
translation, the main pathway of ribosome recycling does not depend on
scanning-like movement through the 3'-UTR. Changes in termination kinetics or
disruption of the Pab1 – eIF4F interaction do not affect recycling, yet the
maintenance of normal in vivo mRNP structure is important to this process.
Using bicistronic ACT1-LUC constructs, elongating yeast ribosomes were found
to maintain the competence to (re)initiate over only short distances. Thus, as
the first ORF to be translated is progressively truncated, reinitiation downstream
of an uORF of 105 nucleotides is found to be just detectable, and increases
markedly in efficiency as uORF length is reduced to 15 nucleotides.
Experiments using a strain mutated in the Cca1 nucleotidyltransferase suggest
that the uORF length-dependence of changes in reinitiation competence is
affected by peptide elongation kinetics, but that ORF length per se may also be
relevant. Thus, the loss of the reinitiation potential of elongating ribosomes
appears to render these incapable to promote intramolecular recycling via
scanning of the 3'-UTR.
5
I. Contents
I. TABLE OF CONTENT
ACKNOWLEDGEMENTS....................................................................................................................... 4 ABSTRACT ................................................................................................................................................ 5 I. TABLE OF CONTENT.......................................................................................................................... 6 II. ABBREVIATIONS ............................................................................................................................... 8 III. LIST OF FIGURES AND TABLES................................................................................................... 9 1. INTRODUCTION ................................................................................................................................ 10
1.1 CONTROLLING GENE EXPRESSION IN YEAST: FROM TRANSCRIPTION TERMINATION TO TRANSLATION INITIATION ................................................................................................................ 11 1.1.1. Pre-mRNA processing........................................................................................................... 11 1.1.2 Translation in the nucleus?..................................................................................................... 13 1.1.3 Transcript export from the nucleus ........................................................................................ 14
1.2 FACTORS AND MECHANISMS IN EUKARYOTIC TRANSLATION ........................................................... 15 1.2.1 Translation initiation .............................................................................................................. 15
1.2.1.1 Recruitment of the 43S preinitiation complex to the 5'-cap ........................................................... 16 1.2.1.2 Selection of the translational start site and peptide synthesis initiation ........................................ 18
1.2.2 Elongation .............................................................................................................................. 20 1.2.3 Termination............................................................................................................................ 21
1.2.3.1 eRF1: tRNA mimicry and stop codon recognition ......................................................................... 21 1.2.3.2 eRF3: from little known eRF1-binding partner to hub for virtually all translation termination
events ............................................................................................................................................ 23 1.2.4 Elements in the 5'-UTR affecting translational efficiency ..................................................... 25
1.2.4.1 AUG context .................................................................................................................................. 25 1.2.4.2 Leader length................................................................................................................................. 27 1.2.4.3 Upstream AUGs and ORFs ........................................................................................................... 27 1.2.4.4 Secondary structures and mRNA-protein interactions................................................................... 28
1.2.5 The circular-loop model of eukaryotic mRNA ...................................................................... 32 1.2.5.1 The mRNA 5' – 3' interaction......................................................................................................... 32 1.2.5.2 The 3'-UTR, a loop within the loop?.............................................................................................. 35
1.3 MRNA STABILITY IN EUKARYOTES.................................................................................................. 35 1.3.1 'Regular' pathways of mRNA turnover in yeast ..................................................................... 36
1.3.1.1 The major pathway of mRNA decay: deadenylation, decapping and 5' 3’ exonucleolytic degradation................................................................................................................................... 36
1.3.1.2 3' 5' exonucleolytic and endonucleolytic mRNA degradation ..................................................... 40 1.3.2 mRNA surveillance and nonsense-mediated decay................................................................ 41
1.3.2.1 The need for surveillance .............................................................................................................. 41 1.3.2.2 Cis- and trans-acting factors in NMD ........................................................................................... 42 1.3.2.3 mRNA surveillance by mRNP (re)organisation ? .......................................................................... 45 1.3.2.4 The evidence for mRNA surveillance in the nucleus...................................................................... 46
1.4 UORFS: REINITIATION OF TRANSLATION VS. RIBOSOME RELEASE ................................................... 48 1.4.1 Polycistronic transcripts in eukaryotes? ................................................................................. 48 1.4.2 uORF control in GCN4 translation: the stop codon context and eIF2 phosphorylation ......... 49 1.4.3 Reinitiation is affected by the intercistronic distance and the uORF length........................... 50 1.4.4 Other factors governing reinitiation ....................................................................................... 52 1.4.5 Reinitiation of translation in prokaryotes and eukaryotes ...................................................... 53 1.4.6 uORFs can regulate transcript stability .................................................................................. 54
1.5 OBJECTIVES..................................................................................................................................... 56 2. MATERIALS AND METHODS......................................................................................................... 57
2.1 CHEMICALS AND ENZYMES.............................................................................................................. 57 2.2 CELL METHODS................................................................................................................................ 58
2.2.1 Strains and cell culturing........................................................................................................ 58 2.2.2 Escherichia coli transformation ............................................................................................. 59 2.2.3 Saccharomyces cerevisiae transformation ............................................................................. 60
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I. Contents
2.3 RECOMBINANT DNA METHODS....................................................................................................... 61 2.3.1 DNA manipulation ................................................................................................................. 61 2.3.2 Screening of E. coli transformants ......................................................................................... 61 2.3.3 CsCl discontinuous gradient DNA purification ..................................................................... 62 2.3.4 Plasmid constructs.................................................................................................................. 62
2.4 RNA METHODS ............................................................................................................................... 67 2.4.1 Total yeast RNA extraction.................................................................................................... 67 2.4.2 Northern blot .......................................................................................................................... 67 2.4.3 RT-PCR.................................................................................................................................. 68 2.4.4 poly(A) length determination by RNase H treatment............................................................. 69
2.5 PROTEIN METHODS .......................................................................................................................... 69 2.5.1 Luciferase assay and protein quantification ........................................................................... 69 2.5.2 Western blotting ..................................................................................................................... 70
2.6 POLYSOMAL GRADIENT ANALYSIS................................................................................................... 71 2.7 IN VITRO TRANSLATION IN CELL-FREE YEAST EXTRACTS.................................................................. 71 2.8 REPRODUCIBILITY ........................................................................................................................... 73
3. RESULTS.............................................................................................................................................. 74 3.1 RIBOSOME RECYCLING: 3'-UTR ACCESSIBILITY AND TRANSLATIONAL EFFICIENCY........................ 74
3.1.1 The potential role of 3'-UTR accessibility for ribosomal recycling on a circular mRNA...... 74 3.1.2 Structures in the 3'-UTR can reduce translational efficiency ................................................. 74 3.1.3 Recruitment of IRP1 to the 3'-UTR modulates translation and not polyadenylation ............. 79
3.1.3.1 poly(A)-length and -site verification.............................................................................................. 79 3.1.3.2 Polysomal analysis ........................................................................................................................ 81
3.1.4 Translation of a full-length ORF is insensitive to changes of the codon context ................... 83 3.1.5 The eIF4G – Pab1p interaction is not required for 3'-UTR-confered translation repression
in vivo .................................................................................................................................... 85 3.1.5.1 The absence of the cap – poly(A) interaction in vivo does not abolish the translational
inhibition by a 3'-UTR structure ................................................................................................... 85 3.1.5.2 In vitro translation in cell-free extracts is not affected by IRP1-binding to the 3'-UTR ................ 85
3.2 CORRELATION OF ORF LENGTH AND RIBOSOME REINITIATION POTENTIAL ..................................... 87 3.2.1 Reinitiation capability increases as a wild type ORF is progressively truncated ................... 87 3.2.2 Translation of a downstream ORF by reinitiation and leaky scanning can be distinguished
using start codon mutants ...................................................................................................... 91 3.2.3 Reinitiation competence is affected by elongation rate.......................................................... 94 3.2.4 Premature translation termination by either ORF truncation or stop codon insertion affects
mRNA stability differently .................................................................................................... 97 3.2.4.1 The presence of premature termination codons but not 3'-truncation of the ORF trigger
nonsense-mediated decay.............................................................................................................. 97 3.2.4.2 PTC-containing mRNAs are stabilised by a full-length downstream ORF that is not translated .. 99
4. DISCUSSION...................................................................................................................................... 101 4.1 ACCESSIBILITY OF THE 3'-UTR IS NOT A PREREQUISITE FOR POSTTERMINATION RIBOSOME
RECYCLING .................................................................................................................................... 101 4.1.1 Differences between 5'-UTR and 3'-UTR directed blockage ............................................... 101 4.1.2 Translation might be affected by a 3'UTR-conferred mRNP modification.......................... 103 4.1.3 Recycling is unlikely to be reinitiation-like since the ribosomal reinitiation potential
depends on the ORF length.................................................................................................. 105 4.2 THE RELATIONSHIP BETWEEN ORF LENGTH, NONSENSE-MEDIATED DECAY AND REINITIATION.... 106
4.2.1 PTC-insertion but not ORF 3'-truncation results in accelerated mRNA decay .................... 106 4.2.2 Insertion of an untranslated downstream ORF stabilises PTC-carrying mRNAs................. 107 4.2.3 Modulation of leaky scanning and mRNA stability by the efficiency of uORF recognition107
4.3 FUTURE PERSPECTIVES .................................................................................................................. 110 5. REFERENCES ................................................................................................................................... 112
7
II. Abbreviations
II. ABBREVIATIONS
Abbreviations used frequently throughout the text are listed below, for
abbreviations of chemicals and enzymes see Tables 2.1 and Table 2.2 in
Section 2.1.
Cap ..... m7Gppp-R structure at mRNA 5'-end CBC .... nuclear cap-binding complex (e)EF... (eukaryotic) translation elongation factor (e)IF .... (eukaryotic) translation initiation factor (e)RF... (eukaryotic) polypeptide chain release
factor hnRNP heterogeneous nuclear ribonucleoprotein IRE...... iron responsive element IRES ... internal ribosome entry site IRP...... IRE-binding protein mRNP . messenger ribonucleoprotein NMD.... nonsense-mediated decay NPC .... nuclear pore complex OD(x) ... optical density (absorbance) at x
nanometres Pab1p . poly(A)-binding protein Pol II ... DNA-dependent RNA polymerase II PTC..... premature termination codon RNP .... ribonucleoprotein
snRNP small nuclear ribonucleoprotein (u)ORF (upstream) open reading frame Upf ..... up-frameshift Amino acid three letter code Ade..... adenine Arg ..... argenine His ...... histidine Ile ....... Isoleucine Leu ..... leucine Lys ..... lysine phe ..... phenylalanine Ser...... Serine Thr...... threonine Trp...... tryptophan Tyr ...... tyrosine Ura ..... uracil Val ...... valine
8
III.List of Figures and Tables
III. LIST OF FIGURES AND TABLES Figure 1.1 The pathway of eukaryotic mRNA from the nucleus to the sites
of translation and decay in the cytoplasm ................................... 12 Figure 1.2 Steps of translation in Saccharomyces cerevisiae ...................... 17 Figure 1.3 Mechanisms of eukaryotic start codon recognition...................... 26 Figure 1.4 Deadenylation-dependent pathways of mRNA degradation in
S. cerevisiae................................................................................ 37 Figure 1.5 Nonsense-mediated decay of premature termination codon
containing mRNAs....................................................................... 43 Figure 3.1 Repression of translation via IRP1-binding to the 3'-UTR. .......... 76 Figure 3.2 Position-dependent decrease of translational efficiency by a
3'-UTR-located poly(G) tract........................................................ 79 Figure 3.3 Binding of IRP1 to an IRE located in the 3'-UTR does not
affect polyadenylation.................................................................. 80 Figure 3.4 IRP1-binding to IREs in the 5' or 3'-UTR affects the polysomal
distribution of LUC mRNA ........................................................... 82 Figure 3.5 The stop codon context of the full-length luciferase ORF does
not affect translational efficiency or mRNA stability..................... 83 Figure 3.6 Significance of 5' – 3' interactions to recycling ............................ 86 Figure 3.7 Truncation of the first ORF in a bicistronic ACT-LUC construct
leads to increased expression of the downstream ORF.............. 89 Figure 3.8 3'-truncation of an actin ORF to 63 nt or shorter triggers decay
of the monocistronic mRNA......................................................... 90 Figure 3.9 Elements that modulate reinitiation affect the low luciferase
activity displayed by the full-length actin-luciferase mRNA. ........ 91 Figure 3.10 Manipulation of the FLAG-ACT mini-ORF upstream of LUC ....... 93 Figure 3.11 Inhibition of translation elongation affects expression of a
downstream luciferase ORF in a uORF-size dependent manner ........................................................................................ 96
Figure 3.12 Impact of a premature stop codon on the translation and abundance of monocistronic ACT and bicistronic the ACT-LUC mRNA.......................................................................................... 98
Figure 4.1 Channelling is promoted by mRNP structure ............................ 104 Figure 4.2 5'-UTR sequence of ACTd105-LUC and representation of
ORFs starting at all possible upstream ATGs. .......................... 108 Figure 4.3 A ribosome flow model for the inhibition of downstream
(re)initiation by uORFs comprising uAUGs................................ 109 Table 2.1 Chemicals used........................................................................... 57 Table 2.2 Enzymes and antibodies. ............................................................ 58 Table 2.3 Correlation of rpm and RCF values in centrifuges and rotors
used. ........................................................................................... 58 Table 2.4 Sequences of oligodeoxyribonucleotides .................................... 63 Table 2.5 Sequences of FL-derived UTRs and ATG-mutated truncated
actin-luciferase constructs........................................................... 64
9
1. Introduction
1. INTRODUCTION Gene expression is the realisation of the information potential encoded in the
genome. While much of its regulation takes place before or at the stage of
transcription, the number and complexity of steps thereafter – mRNA
processing, translation and protein localisation – adds significantly to the
diversity and regulation of gene expression. The scheme in Figure 1.1 outlines
the fate of eukaryotic transcripts following transcription and illustrates that
mRNA is almost continuously processed, protected, transported, translated or
degraded, mostly within specialised cellular compartments. As a consequence,
mRNA is usually not present in the cell without RNA-binding proteins, a fact that
is reflected by the more appropriate use of the term mRNP for the protein-
associated transcript.
The complexity of gene expression in eukaryotes is also mirrored in the
variety of ways that control it; theoretically every step from transcription to
protein degradation can be used to manage the way and extent to which certain
genetic information is utilised. Therefore, in this context and throughout this
study, the characterisation of a step as 'rate limiting' should be approached
carefully since it can be misleading and meaningless. Although some steps
such as initiation of translation feature predominately in the control of
posttranscriptional gene expression, most stages contribute not excessively
unequally to maintain a possibility for balanced regulation at each step along a
pathway (compare also McCarthy, 1998).
Keeping this in mind, this study focuses on a historically somewhat
neglected step in posttranscriptional gene expression, the termination of
translation. Evidence has accumulated that this step is not only the end of
protein synthesis but also a very important regulation site for mRNA-specific
translational efficiency and transcript stability. Besides, very little is known about
the fate of the posttermination ribosome and how it is recycled to start another
round of translation. This study aims to fill this gap by utilising both a structural
approach whereby the mRNA is seen as a circular entity and a functional
assessment of the reinitiation potential of posttermination ribosomes.
10
1. Introduction
1.1 Controlling gene expression in yeast: from transcription termination to translation initiation
1.1.1. Pre-mRNA processing Processing of posttranscriptional pre-mRNA takes place in the eukaryotic
nucleus and bestows transcripts with features important for subsequent
translation, e.g. a 5'-cap structure, an intron-free open reading frame (ORF) and
a precisely defined, polyadenylated 3'-end (Figure 1.1).
Capping
Capping is the addition of GMP to the 5'-triphosphate end of the transcript in the
unusual 5'–5' direction and the subsequent methylation of position N7 of the
transferred GMP. This process is required for cell viability in Saccharomyces
cerevisiae (Shatkin, 1976; Mao et al., 1996) due to its resulting protection of
mRNAs from 5' 3' exonuclease activity (Furuichi et al., 1977; Caponigro &
Parker, 1996). In the nucleus, the m7G cap is bound by the heterodimeric cap-
binding complex CBC, which contains two proteins, CBC20 and CBC80,
respectively. Upon export through the nuclear pore complex, the nuclear
binding proteins are exchanged for the cytoplasmic translation initiation factor
eIF4E (Shatkin & Manley, 2000, for review). In the cytoplasm, cap-binding of
eIF4E is inhibiting recruitment of the decapping enzyme Dcp1p to the
transcript's 5'-end and subsequent mRNA degradation (Vilela et al., 2000;
Ramirez et al., 2002). Recruitment of eIF4E and concurrently of other
translation initiation factors to the cap also enhances translation by promoting
the engagement of the ribosomal subunits with the mRNA, linking thereby
mRNA stability with translation initiation. This is achieved partly by the
interaction of the cap-binding complex with the poly(A)-binding protein (Pab1p
in yeast, PABP in higher eukaryotes; see Section 1.2.5 for details).
A theme continuously revisited in this study is the view that processes
such as pre-mRNA modification are not a sequence of isolated steps, although
they can be separated into distinct, successive stages. As reviewed by
Proudfoot (2000) and Maniatis & Reed (2002), one of the most significant
conceptual shifts in present studies of gene expression is to focus on how
different stages interconnect. For example, RNA-processing factors appear to
substitute the original transcription initiation factors on the transcribing RNA
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1. Introduction
polymerase II (Pol II). Thus, by 'riding along' with the Pol II elongation complex
as it makes the nascent RNA, they couple transcription with mRNA processing.
For example, capping of transcripts is catalysed by enzymes binding to a C-
terminal domain of Pol II, affecting both 5'-end methylation and transcription
elongation (reviewed by Neugebauer & Roth, 1997). This 'coupling' or 'looping
nuclear membrane
transcription initiation
Pol II
splicing
5´
DNA
promoter
Exon
1
pre-mRNA
Exon
2
Intron
AAUAAA
AGAU
capping
p-R-ppp-m7G
transcription elongation
transcription termination
RNA cleavage
3´-end processing
polyadenylation
cap5´UTR 3´UTRORF
+ An
mRNAcircularisation (?)
localisation (?)
mRNP export
factor bindinghnRNPs, export factors
CBC exchanged for eIF4E
nuclear translation
nonsense-mediated decay (NMD) ][ ?“pioneering round“
eIFs, eEFs, eRFs etc
decay
translation
regulatory factorsRNases
NPC
nuclear membrane
transcription initiation
Pol II
splicing
5´
DNA
promoter
Exon
1
pre-mRNA
Exon
2
Intron
AAUAAA
AGAU
capping
p-R-ppp-m7G
transcription elongation
transcription termination
RNA cleavage
3´-end processing
polyadenylation
cap5´UTR 3´UTRORF
+ Ancap5´UTR 3´UTRORF
+ An
mRNAcircularisation (?)
localisation (?)
mRNP export
factor bindinghnRNPs, export factors
CBC exchanged for eIF4E
nuclear translation
nonsense-mediated decay (NMD) ][ ?“pioneering round“
eIFs, eEFs, eRFs etc
decay
translation
regulatory factorsRNases
NPC
Figure 1.1. The pathway of eukaryotic mRNA from the nucleus to the sites of translationand decay in the cytoplasm. Transcription and mRNA processing in the nucleus are concertedmechanisms, governed by RNA polymerase II. The concept of nuclear translation of theprocessed mRNA and coupled nonsense-mediated decay of the transcript in the nucleus arecurrently under discussion. Modified from McCarthy & Kollmus (1995) and Proudfoot (2000), fordetails see text. pol II = DNA-dependent RNA polymerase II, R = purine (at this position in yeastwith 75% relative frequency an adenine, with 25% a guanine), AG/AU = dinucleotides definingthe exon boundaries, AAUAAA = positioning element for 3' RNA cleavage and polyadenylation,ORF = open reading frame, UTR = untranslated region, CBC = heterodimeric nuclear cap-binding complex, eIF4E = mostly cytoplasmic cap binding eukaryotic translation initiation factor,NPC = nuclear pore complex.
12
1. Introduction
back' of processes will be revisited when the links between translation and
mRNA decay and eventually, translation initiation and termination on a circular
mRNA are discussed.
Splicing
The next step to take place on the nascent transcript is intronic splicing.
However, there are only few intron-containing pre-mRNAs in the budding yeast,
and most of them do have only one small intron near the beginning of the
transcript (Sakurai et al., 2002). They are removed in the nucleus due to the
activity of the spliceosome, a macromolecular machine containing five small
nuclear RNAs and numerous proteins (at least 145 in the human spliceosome)
rivalling the ribosome in its complexity (Zhou et al., 2002). Whether splicing in
S. cerevisiae occurs co-transcriptionally has not yet been shown. However,
there is a tight association of these two processes in mammals (reviewed by
Neugebauer & Roth, 1997), and all posttranscriptional processing events are
influencing each other's efficiency and specificity (reviewed by Proudfoot et al.,
2002).
3'-end processing
The final alteration of nuclear pre-mRNA is 3'-cleavage and addition of a poly(A)
tail (Zhao et al., 1999). As discussed more thoroughly in Section 1.2.5, the
poly(A) tail, by providing a binding site for Pab1p, enhances the translation and
stability of mRNA (Sachs et al., 1997). Like capping and splicing, 3'-cleavage
and polyadenylation factors have also been clearly associated with the C-
terminal domain (CTD) of the transcriptionally elongating Pol II (Barilla et al.,
2001). In yeast, the termination of RNA polymerisation has been shown to
depend not only on a RNA signal but also on 3'-cleavage factors (reviewed by
Neugebauer & Roth, 1997).
1.1.2 Translation in the nucleus? Recently, evidence has accumulated to point at a translation-like scanning of
transcripts in the nucleus. Although indicated as early as 1978 by Goidl and
colleagues, more compelling evidence has been put forward by Fortes et al. in
2000 showing that the nuclear cap binding complex CBC binds to both the cap
structure and translation initiation factor eIF4G, mediating thereby translation
initiation. Moreover, Iborra and colleagues (2001) localised the sites of
13
1. Introduction
translation in permeabilised mammalian cells and found that 10-15% of the
labelled tRNA was incorporated in the nucleic fraction, challenging thereby the
long-established theory of compartmentalisation of transcription and translation
in eukaryotes. Furthermore, their findings indicate that nucleic translation is
coupled to transcription, and that the sites of translation within the nucleus
coincide with patterns of translation initiation factor eIF4E. The results of this
work have also been commented on by Hentze (2001) and, more critically, by
Dahlberg and colleagues (2003). Weaknesses pointed out include that not all
translation-related factors have been found to be present in the nucleus at
sufficient high levels, but some are even actively exported into the cytoplasm
(Bohnsack et al., 2002). Furthermore, since the nascent ribosomes in the
nucleus differ from their cytoplasmic counterparts, they would be expected to
not be translationally active or at least to translate in a different way. Thus, the
current view of translation in the nucleus is ambiguous, and needs further
clarification. However, this model could explain observations of degradation of
transcripts via the nonsense-mediated decay pathway protecting the cell from
the export of aberrant transcript to the sites of quantitative translation in the
cytoplasm. Details of the mRNA surveillance mechanism and the consequences
of these findings are discussed in Section 1.3.2.
1.1.3 Transcript export from the nucleus The next step in mRNA utilisation – translation – requires its transport from the
nucleus to the cytoplasm through the nuclear pore complexes (NPCs, Stoffler et
al., 1999). Prior, mRNPs are bound by heterogeneous nuclear
ribonucleoproteins (hnRNPs), known to shuttle between the two compartments
(Sträßer & Hurt, 1999) although they are not considered to play a direct role in
mRNA export (Zenklusen & Strutz, 2001). Much has been discovered about
nuclear export of mRNAs and its connection with upstream gene expression
steps, yet the complex and diverse routes of mRNA export are still under
investigation (Reed & Hurt, 2002). However, there is clear evidence that nuclear
export is a checkpoint for proper mRNA processing since capping, intron
removal, proper 3'-end processing and polyadenylation are all enhancers of
nuclear mRNP export (Zhao et al., 1999). Following nuclear export, mRNA can
be transported to specific sites with the cells (Farina & Singer, 2002) or even be
dormant for months until needed (Standart & Jackson, 1994).
14
1. Introduction
1.2 Factors and mechanisms in eukaryotic translation Functionally, translation can be divided in all organisms into three phases.
During initiation, the translational apparatus is assembled on the messenger
RNA and recognises the start site. Throughout elongation the actual protein
synthesis takes place, while in the last step, termination, the translation is
ended, the peptide product released and the ribosomal subunits recycled to
start another round of translation. Despite apparent homologies and analogies,
translation in eukaryotes differs fundamentally from the situation in prokaryotes,
e.g. in the way the start codon is recognised, in the size and composition of the
ribosomal subunits, and in the origin and complexity of some translation factors
(McCarthy & Gualerzi, 1990; Pain, 1996; McCarthy, 1998; Kozak, 1999; Preiss
& Hentze, 1999).
1.2.1 Translation initiation Initiation is the first step towards the translation of an mRNA, and due to its
complexity it is also the site of extensive global and transcript-specific regulation
of gene expression (Merrick & Hershey, 1996). Essentially, there are three ways
to initiate translation in eukaryotes: via the 5'-end, by recruiting the ribosome to
an internal ribosome entry site (IRES) and by reinitiation of translation by
posttermination ribosomes.
In the IRES mechanism, translation initiation is mediated by a cis-acting
element that recruits the translational machinery to an internal initiation codon
with the help of trans-acting factors (reviewed in Jackson, 2000, and Martínez-
Salas et al., 2001). While common in mammalian and plant viruses, it has only
been recently shown to work in yeast as well (Paz et al., 1999; Zhou et al.,
2001). Moreover, it should be noted that although most researchers in this field
will support the model of internal initiation, there is also an ongoing discussion
about the authenticity of some of the evidence that may result in the reduction
of proposed IRES elements (Kozak, 1992; Kozak, 2001a; see also comment in
Schneider, 2001). In addition, almost all putative IRES elements were detected
either on highly regulated mRNAs with a particularly structured leaders or when
the 5'-end dependent initiation pathway is disrupted in general, e.g. due to
starvation or virus infection (Lamphear et al., 1995; Iizuka et al., 1995; Jackson,
1996). Given that IRESs are capable of promoting internal initiation of
translation on eukaryotic polycistronic mRNAs, it is also striking that this is
15
1. Introduction
never the case (McCarthy, 1998; Kozak, 1999). Therefore, the main
physiological role of IRES-mediated ribosomal entry is apparently to render the
translation of specific ORFs independent of the almost exclusive main initiation
pathway, the recruitment via the 5'-end as in the case of a viral infection of the
cell.
1.2.1.1 Recruitment of the 43S preinitiation complex to the 5'-cap By far the most common route to initiate translation in eukaryotes is the
recruitment of the ribosome to the 5'-cap structure. This is afforded by a bridge
consisting of the cap structure, the cap-binding complex eIF4F, eIF3 and the
40S subunit. Figure 1.2 shows a consensus model since different models for
the order of assembling are currently under discussion (Sachs et al., 1997,
Gingras et al., 1999). The recent report of a reconstituted yeast translation
initiation system might aid in this effort (Algire et al., 2002). In a first step
however, the ribosomal subunits that form under physiological conditions
predominantly 80S complexes have to be separated into 40S and 60S subunits.
This happens under the influence of the factors eIF1A and eIF3, which bind to
the smaller subunit, and eIF6 that binds to the 60S subunit, thereby
sequestering the 80S complexes (Chaudhuri et al., 1999; Si & Maitra, 1999).
The smaller subunit is then charged with the initiator tRNA through binding of an
eIF2-GTP-Met-tRNAi ternary complex (Hinnebusch, 2000). The complete
complex, comprising the 40S subunit stabilised by eIF1A and eIF3 and bound to
the eIF2 ternary complex, is then termed the 43S preinitiation complex (Figure
1.2). Specificity for the mRNA 5'-end is acquired by the group 4 initiation factors,
which are also assumed to facilitate ribosomal binding through removal of
secondary structure on the RNA. Though biochemical evidence for the
described interaction has been obtained, yet again the order of 43S preinitiaion
complex assembly and mRNA recruitment is not fully resolved (Trachsel, 1996).
Certainly, a close association of the 40S subunit and mRNA before the (re-)
binding of the ternary complex can occur as implied by the current model of
reinitiation of translation (see Section 1.4).
eIF4E is the cap-binding component of the initiation factor complex eIF4F
enlisting the largest subunit, eIF4G, a multipurpose adapter capable of
recruiting a number of activities to the 5'-end. The binding of eIF4G to the
ribosome-associated initiation factor eIF3 is thought to establish physical
16
1. Introduction
contact between the mRNA and the 40S ribosomal subunit, whereas binding to
the RNA helicase eIF4A by eIF4G was proposed to be necessary for the
disruption of secondary structures in the 5' untranslated region (Figure 1.2). The
helicase activity of eIF4A is strongly stimulated by the non-essential eIF4B,
however there is no evidence that the proteins physically interact in a stable
manner (Gingras et al., 1999, for review). With the recent finding that eIF4A
associates with eIF4G in yeast (Neff & Sachs, 1999; Dominguez et al., 1999), it
is now agreed that the cap-binding complex eIF4F comprises the translation
initiation factors eIF4E, eIF4G and eIF4A in eukaryotes from mammals to plants
+ An
40S
ternary complex
eIF4F·mRNA
AUGPab1AAAn
40S ribosomal subuniteIF3eIF1A
Met-tRNAi
mRNA
eIF4E·4E-BP
Met-tRNAi·eIF2·GTP
60S
– GTPeIF3 eIF1AeIF2
eIF2·GTP
GTP GDP
eIF2·GDP eIF2B
eIF4E
4E-BP·PO4- eIF4G
eIF4F
40S (Met-tRNAi)– GTPeIF3 eIF1A
eIF2
43S preinitiation complex
48S initiationcomplex
eIF4E
eIF4G
eIF4AeIF4B
scanning
ADP ATP interaction with eIF4G
40SAUG
GTPeIF3 eIF1A
eIF2
eIF4E
eIF4G
?
eIF1A, eIF3,eIF2-GDP+P (?)
eIF540SAUG
polypeptide synthesis initiation
eEFstRNA ( )amino acids ( )
AUG + An
EAP-eEF1A-GTP
eEF2
ATP
ADP+PO4-
eEF1B
GTP GDP+PO4-
UAA
eRFs, Upf1-3p
eEFsAUG
H2Oelongation
ribosome recycling reinitiation
Upf1Upf3 Upf2
Pab1eRF3
eRF1
mRNA decay (NMD)
GTP
40S recruitment
termination
?
+ An
40S40S
ternary complex
eIF4F·mRNA
AUGPab1AAAn
40S ribosomal subuniteIF3eIF1A
Met-tRNAi
mRNA
eIF4E·4E-BP
Met-tRNAi·eIF2·GTP
60S60S
– GTPeIF3eIF3 eIF1AeIF1AeIF2eIF2
eIF2·GTP
GTP GDP
eIF2·GDP eIF2B
eIF4E
4E-BP·PO4- eIF4G
eIF4F
40S40S (Met-tRNAi)– GTPeIF3eIF3 eIF1AeIF1A
eIF2eIF2
43S preinitiation complex
48S initiationcomplex
eIF4E
eIF4GeIF4G
eIF4AeIF4B
scanning
ADP ATP interaction with eIF4G
40SAUG
GTPeIF3eIF3 eIF1AeIF1A
eIF2eIF2
eIF4E
eIF4GeIF4G
?
eIF1A, eIF3,eIF2-GDP+P (?)
eIF540SAUG
polypeptide synthesis initiation
eEFstRNA ( )amino acids ( )
AUG + An
EAP-eEF1A-GTP
eEF2
ATP
ADP+PO4-
eEF1B
GTP GDP+PO4-
UAA
eRFs, Upf1-3p
eEFsAUG
H2Oelongation
ribosome recycling reinitiation
Upf1Upf1Upf3Upf3 Upf2Upf2
Pab1Pab1eRF3eRF3
eRF1
eRF1
mRNA decay (NMD)
GTP
40S recruitment
termination
?
Figure 1.2. Steps of translation in Saccharomyces cerevisae. Recycling of eIF2 andformation of ternary complex, competition between eIF4E-BP and eIF4G for eIF4E-binding, andthe formation of the cap-binding complex are outlined. Recruitment of eIF4F via the Pab1 –eIF4G – eIF3 interaction is an alternative route for ribosomal mRNA association. Duringscanning, the helicase/annealing activity of eIF4A/4B (recruited by eIF4G) is thought to resolveRNA structures such as the depicted 5'-UTR stem-loop. The order of 43S preinitiation complexformation and the fate of the eIF4F complex after 40S binding are still under discussion. Studiesof reinitiation revealed that the release and recycling of at least initiation factor eIF2 occursgradually during translation elongation. Modified from McCarthy (1998), see text for details.eIF4E-BP = eIF4E binding protein (p20 in S. cerevisiae), PO4- = inorganic phosphate.
17
1. Introduction
and yeast (Gingras et al., 1999). Initiation factor eIF4G also contains a binding
site for Pab1p, thereby enabling the poly(A) tail to promote ribosome
recruitment to the mRNA's 5'-end independent from the cap structure.
Moreover, 5' and 3'-ends act synergistically to enhance translation rates, the
mechanism of which is thought to be mRNA circularisation due to the eIF4G –
Pab1p interaction (see Section 1.2.5 for details).
In addition to the translation initiation factors, a number of small proteins
compete with eIF4G for binding to eIF4E. Thereby, these eIF4E-binding
proteins (eIF4E-BPs) can regulate the formation of the eIF4E – eIF4G complex;
the following disruption of the cap-binding complex was observed to result in the
inhibition of cap-dependent, but not of internal initiation of translation. In turn,
phosphorylation (regulated via a number of signal transduction pathways) of
these eIF4E-BPs control their ability to bind to eIF4E (Figure 1.2; Proud, 2002,
for review). In S. cerevisiae, a small protein, p20 with an actual size of 18 kDa
co-elutes with cap-complex preparations (Zanchin & McCarthy, 1995). Its role
remains unclear since deletion of the p20 gene is not lethal and does not affect,
or only slightly increases, the growth rate in rich medium under standard
laboratory conditions (de la Cruz et al., 1997). Recent findings point to a role as
a fine regulator of protein synthesis, since p20 binds to a eIF4E site that is
partially shared by eIF4G, thereby modulating the cooperative binding of eIF4F
to the cap (Ptushkina et al., 1998).
1.2.1.2 Selection of the translational start site and peptide synthesis initiation
The prokaryotic ribosome can base pair with sequence elements such as the
Shine-Dalgarno sequence directly, locating thereby the start codon at any
position on the mRNA (Steitz & Jakes, 1975). In contrast, the eukaryotic 40S
subunit, with its associated cohort of initiation factors, is thought to traverse the
mRNA 5'-UTR from the cap in a linear and processive fashion in a 5' to 3'
direction (Figure 1.2; Kozak, 1977; Kozak, 1978; Kozak and Shatkin, 1978;
Kozak, 1989b; Kozak, 2002). This process termed 'scanning' continues until an
AUG in a favourable start codon context is recognised by the Met-tRNAi bound
to the 40S subunit in the 48S initiation complex (Figure 1.2). Initiation factor
eIF5 mediates the joining of the 60S subunit to the 48S initiation complex and
the release of most other factors. eIF5, together with eIF2, also controls the
18
1. Introduction
stringency of AUG selection, which is marked by the hydrolysis of eIF2-bound
GTP (Huang et al., 1997). The dissociation of eIF2, complexed with GDP, and
the recycling by eIF2B will be discussed together with reinitiation of translation
in Section 1.4.2. Finally, eIF5A is involved in formation of the first peptide bond,
thus linking translation initiation and elongation. These events have been
described in detail in several reviews. (McCarthy, 1998; Hershey & Merrick,
2000; Pestova et al., 2001).
Evidence for the scanning mechanism comes from many directions: The
model was first proposed when edeine-treated cells showed a lack of AUG
recognition and subsequently, almost exclusive association of mRNAs with 40S
ribosomal subunits (Kozak & Shatkin, 1978). Furthermore, a structure inserted
between the cap and the AUG codon was shown to effectively interrupt
scanning and prevent translation initiation with the mRNA co-sedimenting with
the 40S fraction (Kozak, 1986; Kozak 1989a). Findings that the cap structure
promotes translation, and that polycistronic mRNAs are almost completely
absent in eukaryotes support the scanning hypothesis (Kozak, 1989b).
Moreover, the ATP-dependence of in vitro scanning indicates that the RNA-
dependent ATPase eIF4A, recruited by the eIF4F complex, might function as
both a helicase to resolve secondary structures and a hypothetical clamp
(Kozak, 1980; Rozen et al., 1990). This was confirmed by the revelation of the
eIF4A crystal structure; the helicase core, i.e., the sequence comprising all
conserved motifs, is composed of two domains forming a cleft in which the
nucleotide will be located (Caruthers et al., 2000).
Yet, despite the overwhelming evidence for 5'-UTR scanning prior
translation initiation, the underlying mechanism still remains to be elucidated. A
main critique point is that movement of the 40S subunit alone on RNA has so
far never been experimentally demonstrated but would constitute a new
property for this macromolecule since elongation of translation is due to the
interplay of both ribosomal subunits (Londei, 1998). The only serious alternative
model for the recognition of the start codon that differs from the linear scanning
mechanism is the utilisation of an internal ribosome entry site (IRES). The
mechanisms, transcript sequences and structural features described in Section
1.2.4 can attenuate steps of translation initiation in yeast. However, they are
mainly based on predicted deviations from the scanning model but do not
19
1. Introduction
provide experimental evidence contradicting the general mechanism of 5'-UTR
scanning and AUG recognition.
1.2.2 Elongation In the process following translation initiation, the coding potential of the ORF is
realised and converted into a peptide sequence. While the ribosome moves
5' 3' in relation to the mRNA, amino acids delivered by aminoacylated tRNAs
are joined by the formation of peptidyl bonds producing a polypeptide (Figure
1.2). This energy-consuming event is promoted by eukaryotic elongation factors
(eEFs) that have been identified in their analogue (and partly homologue)
similarity to the biochemical mechanism of their prokaryotic counterparts. GTP
and aminoacyl-tRNA are recruited to the A-site of the ribosome by the highly
abundant eEF1A, homologue to the prokaryotic EF1A (formerly known as EF-
Tu). Recognition of the codon by the charged tRNA involves the hydrolysis of
GTP bound to the ternary complex; the eEF1A-GDP complex in turn is recycled
by eEF1B (analogous but dissimilar to the prokaryotic EF1B, previously called
EF-Ts). After transfer of the nascent peptide chain onto the aminoacyl-tRNA
localised in the A-site, another G-protein, eEF2 (analogous to EF2 or EF-G,
respectively), is responsible for the translocation of the charged tRNA into the
P-site and the transport of the deacylated tRNA in the E-site. The next round
starts when the A-site is loaded again with a ternary complex. Yeast and fungi
differ from other eukaryotes due to their dependence upon eukaryotic
elongation factor 3 (eEF3, Qin et al., 1990; Triana et al., 1993). eEF3 is
essential in vivo and required for each cycle of the translation elongation
process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-
tRNA, a function performed by eEF1A, by removing deacylated tRNA from the
ribosomal exit (E) site (Anand et al., 2003).
The elongation rate is mainly determined by the amount of aminoacylated
tRNAs available as well as by the activity of various elongation factors. Because
of its processivity – the succession of equal steps – the elongation phase can
be viewed as a single entity. Any change in the rate of elongation would
therefore only alter the ribosome density on the ORF but not impose a rate
limitation on translation in general. An exception to this might be situations
whereby slowed ribosomes 'queue' to terminate or (re)initiate translation. For
example, pseudoknots are thought to delay elongating ribosomes and enable
20
1. Introduction
the ribosome to perform a -1 frameshift at a slippery site, e.g. in yeast double
stranded RNA viruses (Plant et al., 2003). However, Kozak (2001b) showed that
a pseudoknot, although inhibitory to scanning or initiating subunits, can be
readily resolved by the elongating ribosomes causing no decrease in translation
rates.
Recently, the complete three-dimensional structure of the prokaryotic 70S
ribosome and the 40 and 60S eukaryotic subunits has provided new
opportunities to study details of the elongation (see Ramakrishnan, 2002, for
review). Progress has since made to elucidate how translocation works
(Joseph, 2003) and how the nascent peptide can regulate translation (Tenson &
Ehrenberg, 2002). It can be expected that the three-dimensional reference
given by X-ray crystallography and cryo electron microscopy will help to
interpret the biochemical and genetic data as well as to design new
experiments.
1.2.3 Termination In eukaryotes, an inframe stop codon translocated into the ribosomal A-site is
identified following the association of ribosomes with the heterodimeric
polypeptide chain release factor (eRF) comprising eRF1 and eRF3 (Stansfield
et al., 1995; Zhouravleva et al., 1995). eRF1 recognises all three stop codons –
UAA, UAG and UGA – and mediates the hydrolysis of the peptide from the
tRNA in the P site (Figure 1.2, Frolova et al., 1994). eRF3, a GTPase with
homology to eEF1A, stimulates this reactions in a GTP-dependent, codon-
independent manner (Frolova et al., 1996). The mechanism envisaged currently
implies that eRF3 associates with the small ribosomal subunit and recruits
eRF1. The formation of the ternary complex ribosome-eRF3-eRF1 then triggers
GTPase activity upon stop codon recognition (Stansfield et al., 1995).
1.2.3.1 eRF1: tRNA mimicry and stop codon recognition Research on eRF1 has recently focused on the functional anatomy of the factor
as revealed by a variety of techniques (Kisselev et al., 2003, and references
therein). Alignment of structure and function has resulted in the assignment of a
ribosome-binding site, a termination codon recognition site, a peptidyl-tRNA
interaction site and an eRF3-binding site (Frolova et al., 2000). A functionally
active 'core' appears to mimic the structure of a tRNA providing thereby stop
21
1. Introduction
codon specificity in a direct protein-RNA interaction (Song et al., 2000; Bertram
et al., 2000). Contact of eRF1 with both ribosomal subunits would stabilise and
modulate complex formation and stereochemistry (Kisselev et al., 2003). eRF1
shares conserved sequence elements but no extensive homologies to the
prokaryotic counterparts RF1 and RF2. In addition, eRF1 can recognise all
three stop codons whereas RF1 terminates translation at UAG, RF2 at UGA,
and UAA is recognised by both factors (Scolnik et al., 1968). This is in
accordance with observations e.g. that yeast mutants showed an omnipotent
suppression phenotype. It was also shown that eRF1 alone can promote the
release of the elongating polypeptide from the ribosomal P-site when a stop
codon is present in the A-site in vitro (Frolova et al., 1994; Zhouravleva et al.,
1995). However, addition of eRF3 alone blocks this reaction but in turn
enhances the reactivity when it associates with GTP (Zhouravleva et al., 1995).
Stansfield and colleagues (1995) clearly showed that an over-expression of
both eRF1 and eRF3, encoded in S. cerevisiae by SUP45 and SUP35, is
necessary to enhance the efficiency of termination, and the interaction of both
factors appears to ensure to proper recognition of stop codons in vivo.
Yet, the stop codon can also be recognised as an alternative signal, i.e.,
for frame-shifting, read-through or selenocysteine incorporation, referred to as
'recoding' for reprogrammed genetic decoding (reviewed in Gesteland & Atkins,
1996). Recoding requires several regulatory elements to subvert the normal
stop codon recognition, in competition with release factors. Although the only
alternative decoding of triplets in S. cerevisiae occurs in mitochondria (Jukes &
Osawa, 1990) and the incorporation of selenocysteine has not described in this
yeast (Krol, 2002), read-through (Bonetti et al., 1995), +1 and -1 frameshifting
(Farabaugh, 1996) have been observed.
Efficiency of termination is effectively governed by the stop codon context
and variations are deliberately used in animal and plant viruses to facilitate
read-through (Gesteland et al. 1992) or even to arrest ribosomes in a coding
sequence dependent manner (Cao & Geballe, 1996). The use of the three stop
codons in S. cerevisiae is strongly biased with UAA (53.1%) > UGA (26.8%) >
UAG (20.2%). Analyses also revealed a function for the fourth base; the
frequency is then UAAA (18.2%), UAAG (13.5%), UAAU (16.6%), UGAA
(9.9%), UGAU (9.6%), and UAGA (8.4%) (Tate et al., 1996). When only highly
22
1. Introduction
expressed mRNAs were assayed, the bias was even stronger, termination of
translation therefore represents a site for posttranscriptional control. Due to its
coding potential for the nascent peptide, the influences of upstream sequences
are more difficult to assess. Indeed, Mottagui-Tabar et al. (1998) report that
both the P-site tRNA and the -2 amino acid have an influence on the efficiency
of termination. They also observed that basic -2 residues were associated with
higher read-through than acidic ones, which is the reverse to the situation in E.
coli. Bonetti et al. (1995) assumed a similar role for the C-terminal codon. It is
noteworthy that stop codon context and translation termination efficiency affect
the fate of the posttermination ribosome governing the choice between
ribosomal release and reinitiation, which is of special interest when discussing
mRNAs comprising upstream ORFs in Section 1.4.
1.2.3.2 eRF3: from little known eRF1-binding partner to hub for virtually all translation termination events
Deletion of eRF3 is lethal in eukaryotes whereas the only functionally analogue
RF3 in prokaryotes is neither essential nor does it bind to the eRF1
counterparts, RF1 and RF2 (for references see Kisselev and Buckingham,
2000). This points to a more complex role for eRF3 than just modulating eRF1
activity in a GTP-dependent manner. Indeed, eRF3 appears to comprise also
the function of a fourth prokaryotic factor, the essential ribosome recycling
factor RRF. RRF, together with elongation factor EF-2 (EF-G) disassembles the
terminating complex by promoting the translocation of the posttermination
ribosome in prokaryotes. The release of deaminoacylated tRNA is followed by
mRNA and ribosomal subunit dissociation (Hiroshima & Kaji, 1972; Hiroshima &
Kaji, 1973; Hirokowa et al., 2002; see Kaji & Hirokawa, 2000, for review). The
only eukaryotic RRF-like proteins identified so far comprise a mitochondrial
targeting signal (Zhang & Spremulli, 1998). Hence, it can be suspected that
recycling of ribosomes in eukaryotes is a property of the eRF1/eRF3 complex,
since they suffice to perform all the functions of prokaryotic RFs. Alternatively,
Buckingham et al. (1997) proposed that eRF3 alone can recycle both eRFs and
ribosomes, but the biochemical, genetic or structural evidence for this is still
missing.
Another very intriguing recent finding was the elucidation of the so-called
extrachromosomal determinant [PSI+], which was first described in S. cerevisiae
23
1. Introduction
as a modifier of nonsense suppression (Cox, 1965). It was found that the [PSI+]
factor is a product of the SUP35 gene encoding eRF3 (Chernoff et al., 1993;
Doel et al., 1994; Ter-Avanesyan et al., 1994). As well as folding into its native
structure, eRF3 is believed to be capable to adopt a second aberrant
conformation, which manifests as a prion-associated phenotype (Chernoff et al.,
1995; Tuite & Lindquist, 1996). In [PSI+] strains, eRF3 is present both as a
soluble factor and as large intracellular aggregates, resulting from the N-
terminal conferred propensity of the prion conformer to coalesce. Due to their
binding sites for eRF3, eRF1 and Upf1p (see below) could also be sequestered
in the eRF3 particles resulting in a concomitant depletion of these three factors
(Paushkin et al., 1997; Czaplinski et al., 1998). Since overexpression of eRF1
does not relieve the allo-suppressor phenotype of a [PSI+] strain but rather
enhances it, it can be assumed that eRF3 diminution itself is responsible for the
observed inefficient termination and elevated read-through (Derkatch et al.,
1998). The physiological significance of the [PSI+] phenotype appears to be in
the proposed synthesis of normally not produced proteins, i.e., translation of
mRNAs containing a nonsense codon within their reading frame or by-passing a
native stop codon to produce extended proteins (Moffat et al., 1994).
Phenotypically, [PSI+] confers upon cells resistance to heat and ethanol stress
(Eaglestone et al., 1999) and, indeed, at least one heat shock transcription
factor comprising a nonsense codon was found to be synthesised in these cells
(Lindquist et al., 1995). Moreover, the negative effects of uORFs in the 5'-UTR
of stress-related transcription factors such as GCN4, YAP1 and YAP2 might be
overcome by the elevated read-through of termination codons of these uORFs.
Some novel reports have shed some additional light onto the role of eRF3,
although the number of discovered interactions is somewhat staggering
(Mugnier & Tuite, 1999). For example, the finding that the nonsense-mediated
decay factor Upf1p (for up-frameshift) comprises a binding site for both eRF1
and eRF3 links stop codon recognition to mRNA decay (Czaplinski et al., 1998).
The consequences of this tight coupling between translation termination and the
degradation of aberrant transcripts will be discussed in Section 1.3.2. In addition
to binding sites for the ribosome, eRF1 and Upf1p, eRF3 can also associate
with poly(A)-binding protein (PABP in mammals, Hoshino et al., 1999; Pab1p in
yeast, Cosson et al., 2002). The effects on mRNA circularisation, translation
24
1. Introduction
initiation and poly(A) tail conferred mRNA stability will be examined in detail in
Section 1.2.5. Two more proteins, Mtt1p (Czaplinski et al., 2000) and Ittp1
(Urakov et al., 2001) were found to interact with both release factors and
modulate translation termination. Their relationship to transcription factors or
DNA-dependent helicases indicates a coupling of termination with other cellular
processes such as transcription and DNA replication, but these speculations
need further clarification. To add to this growing list, there are also recent
reports of an involvement of the release factors in cytoskeleton organisation and
cell cycle regulation (Valouev et al., 2002).
Hence, eRF3 obviously plays many roles in termination: GTP-dependent
interaction with the ribosome and eRF1, proofreading function, ribosome
recycling and binding-site for Pab1p, Upf1p and other proteins. This reinforces
the status of translation termination as an important control site coupling,
among others, proper stop codon recognition, efficiency of translation
termination, mRNA circularisation, mRNA decay, ribosome release and
reinitiation of translation.
1.2.4 Elements in the 5'-UTR affecting translational efficiency
1.2.4.1 AUG context Initiation of translation in eukaryotes is usually restricted to the AUG start
codon, and the surrounding sequence affects the efficiency of its recognition. In
higher eukaryotes, derivation from the ideal consensus sequence, 5'-
GCC(A/G)CCAUGG-3', leads to significantly decreased translation initiation
efficiency, especially when the two underlined bases (a purine in position -3 and
a G in position +4) are mutated (Kozak, 1987b). However, the effects of
changes are considerably smaller in S. cerevisiae than in vertebrate systems.
An analysis of 131 genes found the bias in nucleotide distribution to be 5'-
A(A/U)AAUAAUGUCU-3' (Cigan and Donahue, 1987). More recently, an index
(AUGCAI) to measure optimal AUG context has been established by Miyasaka
(1999) stating the slightly different optimal consensus sequence 5'-
(A/U)A(A/C)AA(A/C)AUGUC(U/C)-3'. A change in the most important position -3
(underlined) results in only a two-fold decrease of initiation efficiency (Cigan et
al., 1988). It is most likely due to this insensitivity that start codons other than
25
1. Introduction
AUG are hardly recognised in S. cerevisiae (Clements et al., 1988; Huang et al.,
1997; Nett et al., 2001).
AUG priority rule
The main evidence for an unidirectional 5' 3' movement of the scanning
complex derives from the observation that the first AUG is almost exclusively
utilised for initiation of translation (Kozak, 1999). Furthermore, even the
numerous cases where the first AUG does not constitute the site of translation
initiation can be explained based on the scanning model.
Leaky scanning
Non-recognition of start codons leads to 'leaky scanning' whereby the ribosomal
unstructured leader
structured leader
reinitiation
leaky scanning
shunting
IRES
An
An
An
An
An
An
IRES
unstructured leader
structured leader
reinitiation
leaky scanning
shunting
IRES
AnAn
AnAn
AnAn
AnAn
An
AnAn
IRES
Figure 1.3. Mechanisms of eukaryotic start codon recognition. Comparison of AUGidentifications mediated by scanning of the 5'-UTR or by recruitment via an internal ribosomeentry sites (IRES). An unstructured leader poses no hindrance to scanning 40S ribosomalsubunit while a sufficiently stable secondary structure can block 5'-UTR scanning. Under certaincircumstances, ribosomes can reinitiate translation having terminated at an upstream ORF(uORF), albeit with usually lower efficiency. In 'leaky scanning' an uORF can be bypassed whenits start codon is in an unfavourable sequence context, the ribosome then initiates at adownstream AUG. The translation of an uORF can also lead to negotiation of an otherwiseimpeding secondary structure by non-linear ribosome 'shunting' (bent arrow), followed byscanning to reach the downstream ORF. Internal initiation of translation does not involvescanning but recruitment of the ribosome via an IRES element to mRNAs that predominantlycomprise highly structured leaders. The 40S ribosomal subunits are depicted as spheres at sitesof translation initiation or scanning blockage. The 5' 3' scanning movement is indicated bystraight arrows, secondary structures are shown as stem-loops, ORFs as boxes.
26
1. Introduction
subunit continues to proceed along the mRNA until it initiates at a start codon
farther downstream (Figure 1.3; Kozak, 1991c and 1999). This mechanism is
widely used in mammalian cells and viruses to produce two different proteins
from the same mRNA, indicating that this mechanism is not 'sloppiness' on the
part of the translational apparatus but employed deliberately (Kozak, 2002, for a
review). The utilisation of start codons within an unfavourable codex can be
enhanced when a downstream secondary structure slows scanning and thereby
provides more time for codon / anticodon recognition. This suppression of leaky
scanning requires a critical distance of 13 –15 nt (corresponding to half the
diameter of a ribosome) between the start codon and the structure (Kozak,
1990).
1.2.4.2 Leader length Recognition of the first AUG requires a certain distance between the mRNA's 5'-
end and the start codon. In in vitro experiments, translation initiation at an AUG
located up to 12 nt from the cap was clearly impaired while placing the start
codon 20 nt downstream of the 5'-end enhanced the recognition rate greatly
(Kozak, 1991a; Kozak, 1991b; Kozak, 1991d for review). One explanation
offered for this is that 80S ribosomes poised at the start site prevent 40S
subunits from binding to the 5'-end, implying that the initiation step might be rate
limiting. Alternatively, a certain leader length might be required to facilitate
contact between the mRNA and the 40S subunit. Observations of translation
rates suggest that lengthening an unstructured leader up to 80 nt has a
proportionally stimulating effect on initiation (1991b). Further extension up to
1,800 nt does see only a moderate influence on the translational efficiency in S.
cerevisiae cells whereas in cell lysates translation was reduced to 1-2%
indicating different initiation or scanning requirements in these systems (Karine
Berthelot et al., private communications). Taken together, these results point at
a certain 5'-UTR length that is 'saturated' with scanning 40S subunits, delivering
them in vivo effectively and without great lost to the site of initiation.
1.2.4.3 Upstream AUGs and ORFs A sizeable group of natural mRNAs have upstream AUGs (uAUGs) or complete
upstream open reading frames (uORFs) in their leader sequence (Cigan and
Donahue, 1987). In accordance with the scanning model, initiation at these
27
1. Introduction
upstream sites reduces translation of downstream ORFs, albeit to varying
degrees. The two possibilities for downstream initiation in these cases are leaky
scanning, where a start codon is not recognised or reinitiation, where each start
codon is duly recognised and several ORFs are translated per mRNA (Figure
1.3). With reference to the scanning mechanism and the first-AUG rule, it
should be noted that posttermination ribosomes can scan backwards to only a
very short extent to reinitiate translation upstream of the termination site (Kozak,
2001b). Therefore, uAUGs that are not followed shortly by an in-frame stop
codon effectively inhibit (re)initiation at downstream start codons. The
phenomenon of uORFs and translation reinitiation in eukaryotes is discussed in
context with ribosome release in detail in Section 1.4.
1.2.4.4 Secondary structures and mRNA-protein interactions
5'-UTR structures can block initiation
The ability of secondary structures located 5' of start codons to be an effective
means to restrict translation initiation on a given mRNA affirms the evidence for
the 'scanning models' (Kozak, 1986; Kozak, 1989b; see Kozak, 1991d and
2002, for review). The strength of the inhibitory effects is apparently linked to
the thermodynamic stability of the structure, but there is an obvious difference
between eukaryotic systems towards the sensitivity of scanning. An estimated
folding stability of approximately -50 kcal mol-1 is required in mammalian cells to
inhibit translation by about 90% (Kozak, 1989a). In contrast, the yeast
translation apparatus shows a greater sensitivity to structured leaders (as
discussed above), stem-loops with a stability of -18 kcal mol-1 suffice to achieve
a comparable reduction (Oliveira et al., 1993b; Saggliocco et al., 1993).
Obviously to avoid longer, and therefore potentially structured leaders, in yeast
the length of the 5'-UTR sequence is usually limited to less than 100
nucleotides (Merrick, 1992). Conversely, those mRNAs that contain extended,
strongly structured leaders have been suggested to be translationally restricted
and regulated (Kozak, 1986). Moreover, the preference for adenines in the 5'-
UTR of baker's yeast (Cavener & Ray, 1991) and their reduced ability to form
stable secondary structures (Cigan & Donahue, 1987) might also reflect an
increased sensitivity of its scanning apparatus. Interestingly, in addition to the
thermodynamic stability of stem-loop structures, their effect on scanning
28
1. Introduction
inhibition increases proportionally with their G+C content (Vega Laso et al.,
1993). However, this should not be confused with impeding guanine-stretches,
whereby a sequence of about 18 Gs located in the 5'-UTR can efficiently
decrease translation and mRNA degradation (Muhlrad et al., 1995) due to
guanosine base stacking – a different mechanism (Williamson et al., 1989;
Zimmerman et al., 1975).
Mammalian systems also show a position-dependence of translational
regulation via secondary structures in the 5'-UTR. In rabbit reticulocyte lysates,
a cap-proximal stem-loop with a stability of -30 kcal mol-1 was found to be
inhibitorier than an equivalent structure placed 53 nucleotides further
downstream. This effect can be explained if the cap-proximal structure
interferes with 40S-mRNA association whereas the downstream stem-loop
would block 40S scanning driven by a thermodynamic force large enough to
unwind the structure due to the ATP-consuming activity of initiation factors
eIF4A/4B. In contrast, there is no cap-proximal potentation effect in S.
cerevisiae (Vega Laso et al., 1993; Koloteva et al., 1997, Kozak, 1991d), so that
variations in the position of a 5'-UTR stem-loop hardly alter the effect. One
suggestion put forward by Vega Laso and co-authors (1993) implies that the
focus of rate control in yeast differs from mammalian systems so that the effect
on the scanning mechanism is equivalent to the influence on ribosome binding
to the 5'-cap. These authors also found that secondary structures in in vitro
systems impose a more severe inhibition to scanning ribosomes than they do in
vivo, while simultaneously the position-dependence was lost in cell lysates.
Therefore, it is difficult to postulate a consensus model for the effects of stem-
loops in different eukaryotic systems. However, it should be noted that Kozak
(2002) also suggests that the differences monitored in the in vitro system might
be due to unconventional transcript starts producing mRNAs lacking the
structure or because of different effects on mRNA stability. These issues can be
dealt with by avoiding in vitro systems or including RNA length and stability
controls.
Ribosome shunting
The model of ribosome shunting describes a mechanism whereby major parts
of a complex leader are bypassed and not melted by scanning ribosomes
(Figure 1.3; Ryabova et al., 2002). This strategy is mostly employed by viruses,
29
1. Introduction
enabling the viral RNA to form extensive stem loops that are excluded from the
scanning process. These structures can comprise non-recognised AUGs or
even binding sites for viral proteins (Hemmings-Mieszczak & Hohn, 1999). The
negotiation of this formation usually requires a full cycle of translation on a short
upstream open reading frame (uORF) followed by non-linear ribosome
migration (ribosome shunt) across the structure. The main ORF is then
identified by subsequent linear scanning in a reinitiation-like mechanism (Figure
1.3; Hemmings-Mieszczak et al., 2000). However, these exceptions to the linear
scanning mechanism do not concern the translation of the majority of cellular
mRNAs, but are means to regulate translation initiation on viral mRNAs or on
transcripts encoding tightly controlled proteins such as growth factors or
components of the gene expression apparatus (Kozak, 2001a; Ryabova et al.,
2002). Although linear scanning represents the usual mechanism, the
eukaryotic translation apparatus obviously possesses the flexibility to combine
reinitiation with a short dissociation from the mRNA.
Secondary structures within the ORF
As mentioned above, a surprising positive effect of secondary structures on
translation was reported when these were situated immediately downstream of
an AUG. In this case, the structure seems to slow the scanning movement of
the ribosome, so that a given AUG is recognised more efficiently, even if the
context is normally unfavourable for initiation (Kozak, 1991). However, if the
structure was too stable, e.g. in the case of a pseudoknot, the scanning
ribosome could not target the start codon and translation initiation was impaired.
In contrast, the elongating ribosome has been shown to readily overcome
secondary structures located in the ORF. Kozak (2001) reported no obvious
effect when placing a pseudoknot derived from viral mRNA in the coding
domain of a CAT transcript and assaying its expression in rabbit reticulocyte
lysate. In the original viral transcript, the pseudoknot was shown to cause
ribosomal pausing during elongating, followed by frame-shifting (Somogyi et al.,
1993). Thus, the scanning ribosomal 40S subunit and the elongating 80S
ribosome differ fundamentally in their ability to resolve structured RNA during
their movement along the transcript.
30
1. Introduction
The IRE-IRP1 system
In contrast to the constitutive limitation of translation initiation imposed by
intramolecular RNA-RNA interaction such as stem-loops, the inducible
synthesis or activation of an mRNA-binding repressor protein allows negative
regulation within a defined period. For example, the human spliceosomal
protein U1A and the bacteriophage MS2 coat protein confer translational
repression on mRNAs harbouring the respective recognition site in their leader
sequence (Stripecke et al., 1994). However, the best-characterised system of
this kind in eukaryotes is based on the binding of iron-regulatory proteins (IRP1
and IRP2) to an iron responsive element (IRE) in the 5'-UTRs of mRNAs
encoding ferritin and erythroid 5-aminolevulinic acid synthetase in vertebrate
cells (Aziz & Munro, 1987; Butt et al., 1996; Rouault and Harford, 2000).
Binding is sufficient to block translation of these mRNAs, but this effect is
relieved when iron levels exceed the requirements in the cell (Rouault et al.,
1988). Oliveira and colleagues (1993a) showed that binding of IRP1 to an IRE-
containing leader results in translational repression in S. cerevisiae,
demonstrating thereby that the regulation requires no other mammalian
components other than IRP1 and IRE to function.
Due to the high affinity of IRP1 to IRE (KD = 10-10 to 10-11 M, Oliveira et al.,
1993a) this system compares easily with the stability of stem loops. However,
the upper stem-loop sequence of the IRE element that suffices for IRP1-binding
is not very stable itself (∆G -7.3 kcal mol-1, Oliveira et al., 1993b). Hence, it
imposes almost no constraints on scanning or elongating ribosomes (compare
Oliveira et al., 1993b; Vega Laso et al., 1993) and can therefore serve as an
ideal negative control in experiment investigating the accessibility of mRNA
UTRs. Similar to Kozak's observation with stable stem loops (Kozak, 1986;
Kozak, 1989a), Koloteva and colleagues (1997) observed a strong inhibition of
translation upon IRP1-binding in yeast cells to a construct with the IRE in the 5'-
UTR. In the original construct (IRE-wt) the stem-loop was 9 nt downstream of
the mRNA's 5'-end preventing efficiently binding of the 43S preinitiation
complex to the cap. However, when the IRE was placed distal to the cap, the
preinitiation complex could bind to the mRNA but not proceed due to the
imposed inhibition of 5'-UTR scanning. These features make the inducible IRE-
31
1. Introduction
IRP system highly suitable to block the movement of 40S subunits at a
predetermined site on any given mRNA in yeast cells.
1.2.5 The circular-loop model of eukaryotic mRNA
1.2.5.1 The mRNA 5' – 3' interaction The idea, that ribosomes on a closed-loop mRNA – termed then circular
polysomes – are the most efficient means to achieve expression of a given
transcript dates back a few decades (Philipps, 1965; Baglioni et al., 1969).
Later, electron microscopy revealed circular polysomes on the rough
endoplasmic reticulum, although the observation of linear and 'G' shaped
polysomes indicated that circularisation might either be not permanent or not
affect all mRNA species (Christensen et al., 1987). However, it was not until
genetic and biochemical methods were developed that the molecular basis of
this phenomenon could be explained and interpreted. The discovery that both
mRNA ends are protected by a cap structure and a poly(A)-tail and recruit
eIF4G and Pab1p, respectively, resulted in Tarun & Sachs (1996) reporting a
direct interaction of these factors that is necessary to promote translation
initiation synergistically and to inhibit mRNA degradation. Subsequently, Wells
et al. (1998) demonstrated that an in vitro reconstituted yeast eIF4E – eIF4G –
Pab1p complex could circularise a capped, polyadenylated mRNA. This
communication between the mRNA's 5' and 3'-end appears to be conserved
throughout all eukaryotes (Imataka et al., 1998; Piron et al., 1998; Wakiyama et
al., 2000), although this interaction might not be direct in mammalian cells
(Craig et al., 1998). Since transcript circularisation potentially concerns
numerous aspects of posttranscriptional control, extensive and recent reviews
on this topic are available (Jacobson, 1996; Jacobson & Peltz, 1996; Sachs et
al., 1997; Gallie, 1998; Kozak, 1999; Sachs & Varani, 2000; Sachs, 2000; Groft
& Burley, 2002).
eIF4G and Pab1p synergistically enhance translation initiation
Functionally, the interaction between initiation factor eIF4G that recruits the
ribosome to the mRNA via eIF3 (see also Figure 1.2) and Pab1p offers a
straightforward explanation, how the poly(A)-tail can promote cap-independent
translation initiation (Munroe & Jacobson, 1990; Tarun and Sachs, 1995;
Lamphear et al., 1995; Tarun et al., 1997). Moreover, Pab1p-binding to eIF4G
32
1. Introduction
increases the affinity of the cap-binding complex eIF4F (of which eIF4G is a
subunit) for the 5'-cap structure, resulting in a synergistically enhanced increase
of cap-dependent translation initiation (Sachs & Davis, 1989; Gallie, 1991;
Gerstel et al. 1992; Iizuka et al., 1994; Tarun and Sachs, 1995; Kessler &
Sachs, 1998; Otero et al., 1999). Alternatively, synergy could also base on
enhanced activity such as the ATPase within eIF4A, when all the factors have
bound. Notably, in addition to its effect on eIF4G and, consequently, 40S
recruitment, Pab1p also promotes the 60S subunit joining step (Sachs & Davis,
1989), most likely via Ski2p/Slh1p and eIF5/eIF5B (Searfoss et al., 2001).
Several groups have described that Pab1p exerts its enhancing effect on
translation initiation also in trans, on a poly(A)-deficient mRNA (e.g. Proweller &
Butler, 1996; Otero et al., 1999). Munroe & Jacobson (1990) and more recently,
Borman et al. (2002) reported that exogenous poly(A) stimulated translation via
Pab1p and eIF4G to the same extent as the presence of a poly(A) tail at the
mRNA 3'-end. Pab1p also could exert its effects in translation and stabilisation,
when it was brought to the 3'-end as a fusion protein, independent from the
poly(A) tail (Coller et al., 1998). Yet, in vivo eIF4G and eIF4B exhibit an
synergistic effect on Pab1p binding affinity for poly(A) (Le et al., 1997), and
since poly(A) deficient mRNAs are translated only poorly (Gallie, 1991) the
activity of poly(A) and thus Pab1p is likely to be almost exclusively
intermolecular and not in trans. For the sake of completeness, it should also be
noted that poly(A)-deficient mRNAs are not efficiently exported from the nucleus
(Zhao et al., 1999). Kozak (1991) suggested that in cells recruitment of Pab1p
to the poly(A) tail increases the 'local concentration' of the binding partner
eIF4G and therefore promotes (re)initiation at the polyadenylated mRNA.
mRNA circularisation and stability
The association of the cap-structure with cap-binding proteins and Pab1p-
binding to the poly(A)-tail prevents mRNA degradation directly by blocking the
access of ribonucleases and decapping enzymes to the mRNA's termini. Yet,
on a circular mRNA Pab1p also protects the 5'-cap by enhancing the affinity of
eIF4G for the cap-binding factor eIF4E (Wei et al., 1998; von der Haar et al.,
2000). This would also explain the sequence of the major mRNA degradation
pathway: deadenylation, decapping and 5' 3' exonucleolytic decay (see also
Section 1.3.1.1). Circularisation and the resulting enhancement of translation
33
1. Introduction
initiation by the 3'-end is also a means to test the integrity of RNA prior to
translation; incomplete or partly degraded mRNA would not sustain high
translation efficiency, preventing the production of truncated proteins.
Ribosome recycling via the poly(A) – Pab1p – eIF4G – cap bridge?
While the poly(A)-binding protein facilitates 40S recruitment by the initiation
factors and the subsequent 60S joining step, circularisation of the transcript
theoretically also offers the possibility of posttermination ribosomes being
directly channelled from the 3' to the 5'-end of the same mRNA, thereby
putatively enhancing the specific translational dramatically. Support for this
theory comes from measurements stating that capped transcripts lacking a
poly(A) tail do not enter translation in polysomes at a degree that was expected
if terminating ribosomes were to disassociate from an mRNA and equilibrate
with free subunits prior to re-entry into a polysome (Baglioni et al., 1969). This
suggested a preferential re-recruitment of those ribosomal subunits already
associated with an mRNA.
In a different approach, Novoa and Carrasco (1999) addressed the
question whether translation is divided between a 'pioneering' round that
recruits ribosomes from the cellular pool and the subsequent rounds that rely on
intramolecular recycling. They added poliovirus protease 2A to a pool of
translated mRNAs, cleaving the eIF4GI and II carboxy-terminally from the
eIF4E/PABP binding regions and blocking de novo translation. Under conditions
of near complete eIF4G cleavage, cellular mRNAs continued to be translated
for hours whereas, for instance, a newly transcribed reporter mRNA was very
poorly translated. This suggests that the eIF4E/PABP 'bridge' left by eIF4G
cleavage suffices to maintain a translationally competent mRNA, probably by
helping terminating ribosomes to reinitiate translation. More direct evidence is
provided by spreads of giant polysome mRNA isolated from the salivary glands
of Chironomus species. Due to the length of the growing nascent peptide the
position of the UTRs can be determined. Indeed, three to four ribosomes are
observed bound to what constitutes the 3'-UTR of these mRNAs (see Figure
1A, Kiseleva, 1989 and Figure 2, Francke et al., 1982), pointing at a
posttermination ribosomal scanning process. This could give ribosomes more
'time' to be re-recruited for another round of translation or they could be
channelled directly from the 3'-end to the cap or the site of translation initiation.
34
1. Introduction
One aim of the presented work is to assay this possibility in context with the
reinitiation (and scanning) possibilities of ribosomes having terminated at a
long, wild type ORF.
1.2.5.2 The 3'-UTR, a loop within the loop? The model discussed above assumes that the link potentially 'channelling'
posttermination ribosome to the transcript's 5'-end is formed by the poly(A)-tail,
Pab1p, eIF4G, eIF4E and the cap (see also Figure 1.4). Yet, recently a new
interaction between eukaryotic translation termination factor eRF3 and the
poly(A)-binding protein has been reported, first in mammalian cells (Hoshino et
al., 1999), then in yeast (Uchida et al., 2002). Cosson and colleagues (2002)
also showed that Pab1p acts in translation termination and that this interaction
is involved in the subsequent rounds of translation but not in de novo formation
of the initiation complex. Interestingly, they also demonstrated that eRF3-
binding disrupts the polymerisation of Pab1p, thereby carrying the termination
signal to the poly(A) tail and probably coupling translation termination and
mRNA deadenylation and decay. Thus, in addition to the possibility of
posttermination ribosomes scanning the 3'-UTR another scenario might be the
case, whereby ribosomes or subunits are directly transferred form the point of
translation termination to the 5'-cap. As well as enhancing translational
efficiency, McCarthy (1998) points out that the events at the termination site
would gain in kinetic control on translation upon coupling with the translation
initiation step that figures predominantly in terms of posttranscriptional control.
Structurally, this would result in the 3'-UTR looping out although this might only
occur at a termination event when eRF1/eRF3 are present at the stop codon.
1.3 mRNA stability in eukaryotes Transcript degradation is not simply the end of an mRNA's natural life but an
important factor in the control of gene expression. The translational efficiency of
a certain gene is dependent on its mRNA steady-state abundance, which in turn
is determined mostly by transcription and mRNA degradation rates. This is
reflected by the physical half-lives of transcripts that range in S. cerevisiae from
less than 1 minute to over 60 minutes, or from 100-fold shorter than cellular
generation times to those spanning several cell cycles (Herrick et al., 1990).
This difference in stability was found not to correlate with transcript properties
35
1. Introduction
such as ORF length, codon bias, ribosome density or abundance (Caponigro &
Parker, 1996; Wang et al., 2001b). In contrast, sequences located in the
untranslated regions or within the ORF can determine the stability of a transcript
(see below for details). Moreover, although the decay rate of 'household'
transcripts appears invariable, some mRNAs are regulated in their degradation
behaviour in answer to environmental cues (see references in McCarthy, 1998,
for example).
Most of the recent work in this field has focused on the pathways of
'regular' mRNA degradation and on the surveillance mechanism that protects
the cell from aberrant transcripts by accelerated transcript decay.
1.3.1 'Regular' pathways of mRNA turnover in yeast
1.3.1.1 The major pathway of mRNA decay: deadenylation, decapping and 5' 3’ exonucleolytic degradation
Extensive work by the Parker group on the turnover of the PGK1 mRNA in S.
cerevisiae has revealed a major pathway for the degradation of most cellular
mRNAs in yeast and higher eukaryotes (Muhlrad et al., 1995; Caponigro &
Parker, 1996; Wilusz et al., 2001).
The shortening of the poly(A) tail to oligo-length and the concomitant
release of poly(A) binding protein Pab1p marks the transition from translation-
competent to decay-susceptible mRNA (Figure 1.4). Deadenylation occurs in
two steps with an initial shortening of the poly(A) tail from 70-90 residues to 10-
15 adenosines, followed by a slower removal of the last nucleotides, termed
terminal deadenylation. Although the loss of Pab1p-association with the mRNA
reduces the translational efficiency drastically, it remains unclear whether any of
the two events are rate-limiting in mRNA decay (Caponigro & Parker, 1996). In
mammalians, a major cytoplasmic deadenylase activity has been assigned to
the poly(A)-specific deadenylating nuclease, initially termed DAN, subsequently
designated as poly(A) ribonuclease, PARN. In yeast, the deadenylation is
surprisingly less well characterised. An obvious candidate is the Pab1p-
dependent Pan2/Pan3 (poly(A) nuclease) complex that trims nascent poly(A)
tails in the nucleus. The cytoplasmic activity might be regulated by the
transcription factor complex Ccr4/Caf1 (see Wilusz et al., 2001, and references
therein). Interestingly, deletion of PAB1 in yeast led to a stabilisation of the
36
1. Introduction
poly(A) tails, implying that Pab1p simultaneously protects the poly(A) tail
and recruits the deadenylase to the mRNA's 3'-end (Mitchell & Tollervey, 2000).
Pab1p-bestowed protection is also partly due to mRNA circularisation conferred
by the Pab1p – eIF4G – eIF4E interaction (Figure 1.4), which also explains the
inhibitory effect of Pab1p on the accessibility of the 5'-end to decapping.
Moreover, in mammalian cells, the deadenylase PARN binds to the cap
structure, thereby preventing the access of the decapping enzymes until poly(A)
tail shortening is completed (Wilusz et al., 2001). However, it remains to be
elucidated exactly how the signal from the 3'-end is transferred to the 5'-end,
deadenylation
AAAAAAAAAAAAAAneIF4G
eIF4EeIFsPab1Pab1 Pab1
AREs
STEs
Xrn1
PANDcp1
+/--+ -- + -
-
poly(A) oligo(A)PAN (?), Ccr4p/Caf1p (?)
A10-15
Pab1
A10-15
eIF4E/4G
5' 3' exonuc leolytic decay
A10-15
Dcp1
Rrp4p, Rr41p/Ski6p, Ski2p, Ski3p, Ski8p, Rrp44p (exosome ?)
A10-15m7Gpp
eRF1, eRF3, Upf1-3p
A10-15
decapping
Dcp1p, Dcp2p, Lsm1-8p Vsp16, Mrt1p/Pat1p/Spb10
+ -
processivedegradation
processivedegradation
+ -
3' 5' exonuc leolytic decay
deadenylation
AAAAAAAAAAAAAAneIF4GeIF4G
eIF4EeIFsPab1Pab1Pab1Pab1 Pab1Pab1
AREsAREs
STEsSTEs
Xrn1
Xrn1
PAN
PANDcp1Dcp1
+/--+ -- + -
-
poly(A) oligo(A)PAN (?), Ccr4p/Caf1p (?)
A10-15A10-15
Pab1Pab1
A10-15
eIF4E/4G
5' 3' exonuc leolytic decay
A10-15
Dcp1
Rrp4p, Rr41p/Ski6p, Ski2p, Ski3p, Ski8p, Rrp44p (exosome ?)
A10-15m7Gpp
eRF1, eRF3, Upf1-3p
A10-15
decapping
Dcp1p, Dcp2p, Lsm1-8p Vsp16, Mrt1p/Pat1p/Spb10
+ -
processivedegradation
processivedegradation
+ -
3' 5' exonuc leolytic decay
Figure 1.4. Deadenylation-dependent pathways of mRNA degradation in S. cerevisiae. The transition from translated and protected transcript to degradation-competent mRNA is shown. Pab1p can recruit the major deadenylase (supposedly PAN or Ccr4p/Caf1p) to the poly(A) tail,which is modulated by AREs (A/U rich elements) in the 3'-UTR and by termination factors such as eRFs and Upfs. After shortening of the poly(A) tail and release of Pab1p the major decaypathway involves decapping by Dcp1 and 5' 3' exonucleolytic degradation. A minority of deadenylated mRNA is degraded by 3' 5' exonucleases, putatively involving the exosome and associated factors. Arrows and lines refer to inhibiting (-) and modulating (+/-) interactions.
37
1. Introduction
thereby ensuring the order of deadenylation and decapping.
Once deadenylated, the protective cap structure of the transcript is
removed exposing the transcript to subsequent 5' 3' degradation (Figure 1.4).
Although in vitro the S. cerevisiae enzyme Dcp1p is sufficient for efficient
decapping, the in vivo reaction is more complex. Mrt1p/Pat1p/Sbp10p appears
to be a key player, the protein co-sediments with 40S subunits and polysomal
fractions and possibly recruits the Lsm complex (Sbp8p/Lsm1p and Lsm2-8p)
that in turn interacts with Dcp1p via Dcp2p, a putative nucleoside
diphosphotase (Mitchell & Tollervey, 2000). Pab1p has also been proposed to
interact with Mrt1p, thereby protecting the cap from removal and regulating the
transition from translation to mRNA degradation. Once deadenylated,
transcripts are decapped at different rates whereas the subsequent final
degradation occurs very rapidly. Hence, it can be assumed that removal of the
cap structure is a key control point and rate-limiting step in mRNA turnover
(Caponigro & Parker, 1996). Interestingly, reductions in the rate of cellular
translation elongation also affect mRNA decapping. Treatment of S. cerevisiae
with the elongation inhibitor cycloheximide stabilises many yeast mRNAs; at
least for the PGK1 and MFA2 transcripts this is due to an inhibition of mRNA
decapping (Muhlrad et al., 1995). Since both, translated and untranslated MFA2
transcripts are stabilised, this points at a general mechanism whereby the
inhibition of decapping is a global response to the decrease in translation
elongation (Beelman & Parker, 1994). Moreover, shifting mutant yeast cells
harbouring a temperature-sensitive allele of the gene encoding tRNA nucleotidyl
transferase enzyme CCA1 to the non-permissive temperature results in a
phenotype similar to that of cycloheximide addition. Protein synthesis is
decreased rapidly, ribosome density on mRNAs is enhanced and several
mRNAs are stabilised (Peltz et al., 1992). Although the precise mode of action
is not known, an interesting possibility is that mRNA decapping is regulated in
response to reduced protein production, with increased mRNA stability
compensating partly for the decrease in translation.
Upon removal of the 5' m7GTP structure, the unprotected mRNA is rapidly
degraded by Xrn1p, a major cytoplasmic 5' 3' exonuclease (Figure 1.4).
Mutations in initiation factor eIF5A led to the accumulation of decapped mRNA,
suggesting that accessibility of the 5'-end of the mRNA is also influenced by
38
1. Introduction
translation initiation events after decapping. Moreover, Xrn1p also interacts with
the Lsm proteins, which might engage the exonuclease to the decapped 5'-end
(Mitchell & Tollervey, 2000).
Determinants of mRNA degradation
In higher eukaryotes, the mRNAs of transiently expressed genes, such as early
response genes for lymphokines, transcription factors etc have a very short half
life that correlates well with the presence of adenylate, uridylate-rich (AU-rich)
instability elements (AREs) in the 3'-UTR (Bakheet et al., 2001). AREs can
affect the efficiency of deadenylation and subsequent steps of decay via their
interaction with a plethora of ARE-binding proteins (reviewed in Wilusz et al.,
2001). For example, the overexpression of the ARE-binding protein HuR/HuA
leads to stabilisation of deadenylated, ARE-containing mRNA whereas
conversely in vivo depletion of AUF1/hnRNP D leads to a strong stabilisation of
diverse ARE-containing mRNAs (Mitchell & Tollervey, 2000). Despite intensive
investigation, it remains unclear how AREs and -binding proteins affect the rate
of deadenylation and decay rates. Possibilities are the disruptions of the PABP
– eIF4G or the 5'-cap – eIF4E interaction, thereby providing access for PARN.
In yeast, destabilising elements have been identified throughout the length
of mRNAs, e.g. in the 5'-UTR of the PPR1 and SDH Ip transcripts, or in the
coding region of several genes including MATα1, HIS3, STE3 and SPO13, yet
how these sequence function in mRNA stability still remains unexplained. The
same is true for the instability elements in the 3'-UTR e.g. of HTB1, MFA2, and
STE3 mRNAs (Caponigro & Parker, 1996). These elements are transferable, for
example by creating chimeric mRNAs composed of parts form stable and
instable mRNAs (Heaton et al., 1992). By employing this method, a stabilising
sequence STE has also been proposed to be responsible for the high stability of
PGK1 mRNA with a half-life of about 35 minutes (LaGarnadeur & Parker, 1999).
Details of the STE sequence, the STE-binding protein Pub1p and the
destabilising sequence element DSE are discussed in context with uORF-
related destabilisation in Section 1.4.6. Recently, a yeast transcript, TIF51A,
has been identified whose stability is regulated through its AU-rich 3'-UTR. Both
yeast and mammalian AREs promote deadenylation-dependent decapping in
the yeast system, and the STE/ARE binding protein Pub1p regulates the
stability mediated by the human TNFα ARE (Vasudevan & Peltz, 2001).
39
1. Introduction
In S. cerevisiae, the stability of mRNAs groups is controlled in response to
growth conditions. E.g., five different meiotic mRNAs are stabilised in response
to the shift from vegetative growth in glucose medium to sporulation conditions
in acetate medium. This effect depends on UME2p and UME5p in an unknown
way but obviously does not involve a change in the rate of deadenylation
(McCarthy, 1998). In another example, the SDH2 mRNA encoding the iron
protein of the succinate dehydrogenase complex is unstable and present at low
level when S. cerevisiae is grown with glucose. The glucose-triggered
degradation and changes in translational efficiency of this transcript and of
another example, the SUC2 mRNA, were traced to the 5'-UTR sequence, yet
the precise mechanism remains unclear (de la Cruz et al., 2002).
1.3.1.2 3' 5' exonucleolytic and endonucleolytic mRNA degradation In addition to above described 5' 3' pathway, the yeast PGK1 transcript can
also be degraded in a 3' to 5' direction, although slower than by the 5' to 3'
decay (Figure 1.4). Therefore, this pathway becomes mainly evident in the
absence of the major 5' 3' exonuclease Xrn1p. The 3' 5' exonucleolytic
activity depends on the Ski proteins and the proteins of the exosome (Jacobs et
al., 1998; van Hoof & Parker, 1999), but a mere scavenger function is unlikely
since e.g. the deadenylated MFA2 transcript poses no substrate for this kind of
decay (Caponigro & Parker, 1996). Given that for the majority of yeast mRNAs
analysed so far, decapping is the major fate of deadenylated mRNAs, 3' 5'
exonucleolytic decay could target only particular mRNAs. For example, van
Hoof et al. (2002) and Frischmeyer et al. (2002) reported recently that
ribosomes stalled at the 3'-end of transcripts lacking a stop codon are degraded
in the 3' 5' direction requiring exosome-associated Ski proteins. However, the
situation in mammals could be different, since Wang and Kiledjian (2001)
reported recently that the exosome-associated 3' 5' exonucleolytic activity
contributes significantly to in vivo mRNA decay. They also identified an
exoribonuclease-dependent scavenger decapping activity, and even in yeast at
least one of the both pathways is required for viability since ski2/xrn1 or
ski3/xrn1 double mutants induce synthetic lethality.
The translating ribosome can also arrive at the mRNA’s poly(A) tail when
the in-frame stop codon was not recognised due to a read-through event. In a
model proposed by Vasudevan et al. (2002) translation proceeds then through
40
1. Introduction
the poly(A) tail displacing Pab1p and adding poly-lysine to the C terminus of the
protein product. At the 3’-end of the non-stop RNA, the ribosome stalls and is
only released by interaction of Ski7p with the now empty A site of the ribosome.
Binding of Ski7p results in recruitment of the exosome and dissociation of the
ribosome. The non-stop mRNA is degraded 3' 5' by the exosome and the
protein product may be targeted for proteolysis by the poly-lysine tract.
However, read-through levels of most yeast mRNAs are well below 1.2%
(Bonetti et al., 1995).
mRNA decay can also be triggered by endonucleolytic cleavage, although
in contrast to an abundance of examples in higher eukaryotes (Jacobson &
Peltz, 1996) until now only one mRNA, encoding the ribosomal L2 protein, has
been found to be the target for endonucleases in yeast (Presutti et al., 1995).
1.3.2 mRNA surveillance and nonsense-mediated decay
1.3.2.1 The need for surveillance In all eukaryotes, mRNAs containing premature translation termination codons
(UAA, UGA or UAG) are highly unstable (for reviews, see e.g. Hilleren & Parker,
1999, and Czaplinski et al., 1999). This phenomenon of mRNA surveillance is
usually termed nonsense-mediated decay (NMD) since the majority of
transcripts recognised by this system comprises 'nonsense' premature
termination codons (PTCs, Peltz et al., 1993; Muhlrad & Parker, 1994; for
reviews see Jacobson & Peltz, 2000, and González et al., 1999). For example,
the presence of any of the three stop codons in the first two thirds of the PGK1
ORF reduces the half-life time from normally ~60 to 4 minutes (Hagan et al.,
1995). Although some 'normal' transcripts such as PPR1 and CTF13 are also
degraded via the NMD pathway, it is assumed that degradation of aberrant
transcripts is required to prevent the production of truncated, potentially
dangerous proteins (Czaplinski et al., 1999). Indeed, concomitant with
accelerated mRNA decay, the recognition of a PTC also decreases the
translational efficiency of the transcript (Muhlrad & Parker, 1999b). Yet, it should
be noted that in S. cerevisiae, this surveillance system is dispensable for normal
growth, indicating that stabilisation of mRNAs that are aberrantly produced does
not suffice to kill the cell (Caponigro & Parker, 1996).
41
1. Introduction
1.3.2.2 Cis- and trans-acting factors in NMD In contrast to the major degradation pathway, NMD does not require initial
deadenylation of the aberrant mRNA, but the cap of the polyadenylated mRNA
is removed directly, followed by a rapid 5' 3' degradation step. In yeast, these
activities are performed by the enzymes Dcp1p and Xrn1p that are also active
in the major deadenylation-dependent pathway, (Muhlrad & Parker, 1994;
Beelman et al., 1995). Notably, decapping of NMD substrates does not require
Mrt1p or Lsm1p, suggesting the decapping enzyme is recruited by a different
mechanism (see references in Mitchell & Tollervey, 2000). Logically, the only
situation where the distinction between normal and premature stop codon can
occur, is the translation termination event. Indeed, the activities of the
surveillance system were found to concentrate on the stop codon site involving
regular and additional termination factors (Figure 1.5). During translation
termination, the release factor eRF1 recognises the stop codon, and eRF3
modulates this activity in a GTP-dependent manner, also triggering peptide
release from the ribosome (see Section 1.2.3). In addition, the Upf1, Upf2 and
Upf3 (up-frameshift) proteins have initially been found to associate with the
termination complex and be able to modulate translation read-through of
nonsense-containing transcripts, 'saving' them from NMD (Leeds et al., 1991;
Peltz et al., 1993; Cui et al., 1995; Lee & Culbertson, 1995; Jacobson & Peltz,
2000). Upf1p demonstrates RNA binding, RNA-dependent ATPase and RNA
helicase activity (Czaplinski et al., 1995 and 1999; Weng et al., 1996a,b and
1998, Bhattacharya et al., 2000) and interacts with both, eRF1 and eRF3
(Czaplinski et al., 1998 and 1999). UPF3 encodes a protein with several
nucleus localisation signals and was shown to shuttle between the cytoplasm
and the nucleus (Lee & Culbertson, 1995; Shirley et al., 1998). Upf1p and
Upf3p do not interact directly with each other but both bind to Upf2p, which
explains both the observed complex formation and the similar effects of any
UPF deletion on mRNA decay (He et al., 1997; Atkin et al., 1997). Upf2p might
also play a crucial role in decapping, since a motif search in the
Schizosaccharomyces. pombe and human homologues of Upf2p has identified
so-called eIF4G homology (4GH) domains that span regions of the eIF4G
protein necessary to mediate the closed-loop formation of the mRNA (Mendell
et al., 2000). These data suggest a model in which Upf2p competes with eIF4G
42
1. Introduction
for 4GH domain-mediated interactions, thus disrupting or preventing the
formation of the stable, closed-loop mRNP structure and preparing the access
of Dcp1p.
Interestingly, although only Upf1p binds to eRF1 directly, all Upf proteins
CBC
An AnDSE PTC DSE
Yeast Mammals
PTC
Hrp1p
ALY/REV
AnPTC
CBC
eIF4E
nuclear membrane
eIF4G
A n
PTC
80S
Upf2
eIF4E
Upf1
Upf3
PTC
AAAAAAAAAAn
eIF4G
eIF4E
eRF3
eRF1 Upf2
Upf1Upf3
Pab1 Pab1
eRF3
eRF1 Upf2
Upf1 Upf3
eRF3
eRF1Upf2
Upf1
Upf3
eRF3
Upf2Upf1
Upf3
eRF3
Upf2Upf1
Upf3
AnPTCDcp1
eIF4EXrn1peIF4G
Dcp1
5' 3' exonuleolytic decay
binding ofmarker protein(s)
factor exchange,mRNA export
Initial translation round(nuclear, perinuclear or cytoplasmic ?):Stop codon recognition & Upf recruitment
mRNA degradation
eRFs & Upfs recycling
decapping
exon junction complex (EJC)
Identification of premature termination
Y14
CBCCBC
An AnDSE PTC DSE
Yeast Mammals
PTC
Hrp1p
ALY/REV
AnPTC AnPTC
CBCCBC
eIF4E
nuclear membrane
eIF4GeIF4G
A n
PTC
80S
Upf2Upf2
eIF4E
Upf1Upf1
Upf3Upf3
PTC
AAAAAAAAAAn
eIF4G
eIF4G
eIF4E
eRF3
eRF3
eRF1
eRF1 Upf2Upf2
Upf1Upf1Upf3Upf3
Pab1Pab1 Pab1Pab1
eRF3
eRF3
eRF1
eRF1 Upf2Upf2
Upf1Upf1 Upf3Upf3
eRF3eRF3
eRF1eRF1Upf2Upf2
Upf1Upf1
Upf3Upf3
eRF3eRF3
Upf2Upf2Upf1Upf1
Upf3Upf3
eRF3eRF3
Upf2Upf2Upf1Upf1
Upf3Upf3
AnPTCDcp1Dcp1
eIF4EXrn1peIF4GeIF4G
Dcp1Dcp1
5' 3' exonuleolytic decay
binding ofmarker protein(s)
factor exchange,mRNA export
Initial translation round(nuclear, perinuclear or cytoplasmic ?):Stop codon recognition & Upf recruitment
mRNA degradation
eRFs & Upfs recycling
decapping
exon junction complex (EJC)
Identification of premature termination
Y14
Figure 1.5. Nonsense-mediated decay of premature termination codon containing mRNAs.In both, yeast (downstream elements) and mammalian cells (exon junction complex), markerproteins bind to sites up- and especially downstream of a PTC supposedly facilitating itsrecognition when positioned within a certain distance. The location of the Upf proteins within thecell is indicated, and as a consensus of different models the recognition of premature terminationduring a first round of translation is shown to be perinuclear. Interaction of the termination factorswith the mRNA's 5'-end leads to decapping by Dcp1p although not via the pathway of normaldeadenylation-dependent decapping. Release of eRFs and recycling of Upfs are thought tooccur by mRNP remodelling.
43
1. Introduction
can interact with eRF3, but Upf2p, Upf3p and eRF1 compete with each other for
this interaction (Wang et al., 2001a). Yet, while this points to a sequential
surveillance complex assembly and rearrangement (Figure 1.5), the
intermediates remain to be characterised. Moreover, in yeast, the Upf proteins
are primarily cytoplasmic and polysomal associated (Atkin et al., 1995 and
1997; He & Jacobson, 1995), whereas the localisation and site of action of the
human homologues is currently under discussion (see Section 1.1.2 on nuclear
translation).
In both, mammalian and yeast systems, marker proteins have been
proposed that bind to specific mRNA sites and indicate via their position relative
to the terminating ribosome whether the termination codon is premature or not.
In S. cerevisiae, loosely defined sequence located between PTCs and the 3'-
UTR have been put forward to function as a signal to trigger NMD. These G/C-
rich sequences, termed downstream elements (DSE), are functional in positions
up to 150 nt downstream of the PTC and on mRNAs other from which they have
been isolated (Peltz et al., 1993; Hagan et al., 1995, Zhang et al., 1995; Ruiz-
Echevarria et al., 1996 and 1998). Recent work identified a protein,
Hrp1p/Nab4p that specifically binds to DSEs and is required for destabilisation
of PTC-containing mRNAs by interacting with Upf1p (Gonzalez et al., 2000). As
a result of this interaction, the Upf proteins could potentially identify the
existence of a close downstream DSE, which would label the termination
'premature' and result in accelerated decay. Conversely, a proper termination
complex would be located 3' of all DSEs and associated markers, thereby
preventing NMD. To explain how degradation of transcripts comprising stop
codons that can be recognised as premature, e.g. those containing upstream
ORFs, is prevented, the Peltz group identified a stabilising element (STE) in
yeast that can obviously interfere with NMD. Although a STE-binding protein,
Pub1p, has been purified that is necessary for controlling the activity of the
NMD pathway, the mode of action still remains unclear (Ruiz-Echevarria &
Peltz, 2000; see also Section 1.4.6 about uORFs).
No DSE sequences have been identified in mammalian cells, yet
premature stop codon identification also relies on a bipartite system (Maquat,
2000). During splicing, an exon-junction complex (EJC) is deposited about 20-
24 nt upstream of the splice site. Of the numerous EJC proteins, only Aly/Ref
44
1. Introduction
and Y14 bind to spliced mRNA and are exported to the cytoplasm (Figure 1.5;
Le Hir et al., 2000), but other components of the marker complex could bind to
one of these proteins and be co-transported out of the nucleus. During the initial
round of translation, the elongating ribosome strips these proteins from the
mRNA. In contrast, a ribosome terminating at a PTC could interact via Upf1p
and Upf2p with Upf3p poised at the exon-junction complex, in a manner similar
to the DSE/Hrp1 recognition by the Upf complex in yeast (Kim et al., 2001).
However, the spatial arrangements in mammalian appears to be more relaxed
since a PTC up to 645 nt upstream of the EJC can still trigger NMD (Neu-Yilik et
al., 2001).
1.3.2.3 mRNA surveillance by mRNP (re)organisation ? A current model of eukaryotic mRNA surveillance suggests that a kind of kinetic
proofreading of proper translation termination takes place and is assessed by
mRNP domain organisation (Hilleren & Parker, 1999). As mentioned above and
depicted in Figure 1.5, the (re)arrangement of the mRNP structure is
significantly shaped by a plethora of components such as the cap-binding
complex, translation initiation (and termination) factors, surveillance complex
components such as Upf1-3p, and the poly(A)-binding protein in addition to
other factors such as DSE-, STE-, ARE- and EJC-binding proteins. In case of
translation termination, the authors propose that the hydrolysis of ATP by Upf1p
acts as an 'internal clock' to verify the site of the termination codon. When
translation termination is completed due to proper mRNP domain organisation
before ATP is hydrolysed, then the mRNA is protected. Conversely, if
termination is slow, ATP hydrolysis and mRNP rearrangement changes the fate
of both, the posttermination ribosome and the mRNA. In support of this model,
Upf1p also acts in 'normal' termination, enhancing constitutive stop codon
recognition, termination complex assembly and suppressing nonsense codon
read-through (Weng et al., 1996a). This role of Upf1p depends on its ATPase
activity (Weng et al., 1998). Upf1-3p – although dispensable for normal growth –
are also thought to regulate decapping and exonucleolytic degradation of both
nonsense-containing and wild type mRNAs (He & Jacobson, 2001).
Interestingly, by virtue of its association with Pab1p and the nonsense-mediated
decay factor Upf1p, eRF3 brings together two complexes that are important for
the stability of mRNA. Thus, in this central position, Upf1p-directed ATP
45
1. Introduction
hydrolysis as well as mRNP remodelling might eventually decide between
Pab1p dissociation and successive 'regular' mRNA decay and NMD triggered
by decapping, or mRNA stabilisation and further translation.
According to the Hilleren & Parker (1999) model of NMD by mRNP
rearrangement, it is not the 'improper' end to translation but rather the lack of
proper termination in a suitable mRNP environment that triggers accelerated
decay. Hence, in addition to PTC and uORF-containing mRNAs, also other
mRNPs with a 'distorted' structure are subject to NMD. As discussed briefly,
spliced mRNAs retain an exon junction complex that signals a terminating
ribosome whether a translation termination is premature (He et al., 1993).
Unspliced transcripts are not only very likely to comprise a PTC in the intron
sequences but the lack of the EJC also explains the observation that
transfection constructs bearing at least one intron tend to yield higher
expression levels (e.g. Zhang et al., 1998). This cannot be compensated for by
using exonic 'failsafe' sequences (Neu-Yilik et al., 2001), leading to the
assumption that intron-less mammalian mRNAs may fail to form a proper
terminal mRNP domain and therefore would be subject to mRNA surveillance.
In this context, it is of interest that yeast, where only few transcripts undergo
splicing, the 3'-UTRs are strikingly homogeneous in length, averaging at about
100 nt (Graber et al., 1999). It is possible that, akin to the mammalian model,
the yeast terminating ribosome needs to interact with proteins bound to the 3'-
UTR. Indeed, in yeast, a mutation in CYC1 results in low levels of mRNAs with
extended 3'-ends, and Muhlrad & Parker (1999a) could show that these
aberrant mRNAs are subject to the NMD pathway. Hilleren and Parker suggest
that the significantly altered 3'-terminal mRNP domains of these transcripts are
marking them for degradation.
1.3.2.4 The evidence for mRNA surveillance in the nucleus Currently, a discussion is going on whether nonsense-mediated decay occurs in
the cytoplasm, during nuclear export or in the nucleus itself (for example,
Dahlberg et al., 2003; Hentze, 2001; see also Section 1.1.3). In yeast, the
nuclear-cytoplasmic shuttling of Upf3p implies a role for the nuclear
compartment in NMD (Shirley et al., 1998) but this may be restricted to mark
mRNAs for degradation outside or at the nucleus. Maderazo and co-workers
(2003) also showed recently that at least in yeast, PTC-containing mRNAs that
46
1. Introduction
were exported from the nucleus were readily recognised and degraded in the
cytoplasm upon reconstitution of the NMD pathway. In support of translation or
at least a scanning-like mechanism in the nucleus, Ishigaki et al. (2001)
reported that in mammalian immunoprecipitated PTC-comprising transcripts are
degraded in association with the nucleic cap binding protein CBP80. The
authors proposed a model, whereby the first round of translation is initiated by
the nuclear cap binding complex and serves to scan for aberrant transcripts.
Additional evidence comes from an observed nuclear splicing escape
mechanism that skip PTC-introducing mutations, and that TCR transcripts
comprising nonsense stop codons are downregulated depending on NMD and
translation factors (Wilkinson & Shyu, 2002; Wang et al., 2002). Furthermore,
Iborra and colleagues (2001) reported that mRNA translation indeed could take
place in the nucleus (see Section 1.1.2 for details). Their findings indicate that
the sites of translation within the nucleus coincides with patterns of a subunit of
the proteasome, indicating how truncated protein products from aberrant
mRNAs could be degraded immediately. Since the model of nuclear translation
– apart from other implications e.g. about the origin of the eukaryotic nucleus –
contradicts a prime orthodoxy in eukaryotic molecular biology, the evidence
obviously has to be scrutinised carefully. Dahlberg and colleagues (2003) point
out that with current methods the perinuclear cytoplasm could cofractionate with
the nucleus, making it impossible to distinguish between events in the nucleus
and those coupled with export into the cytoplasm. This possibility of the
'privileged' pioneering round to occur not in the nucleus but during nuclear
export is a main point of critique (see also Figure 1.5). It is probably at this
location the that nuclear cap-binding complex CBC is readily exchanged for
eIF4E that then can interact with Pab1p, resulting in a circularised mRNA.
Subsequent rounds of translations would then take place on a circular mRNA
with the 5' – 3' interaction offering both, protection from degradation and
enhanced translation efficiency.
47
1. Introduction
1.4 uORFs: reinitiation of translation vs. ribosome release
1.4.1 Polycistronic transcripts in eukaryotes? The majority of eukaryotic transcripts are monocistronic in a sense that they
comprise only a single major ORF. However, according to a survey in 1987 by
Kozak, in less then 10% of eukaryotic mRNAs the 5'-untranslated region is not
what it ought to be: untranslated. This mRNA part, hence better referred to as
'leader', can comprise upstream start codons (uAUGs) or upstream open
reading frames (uORFs). Following the proposed mechanism of 5'-end
ribosomal recruitment and subsequent 5' 3' scanning of the leader, these
elements exert a strong regulative role on the initiation frequency at the main
ORF (Geballe, 1996). Fittingly, the majority of uORFs is found in certain classes
of mRNAs involved in the control of cellular growth and differentiation (Kozak,
1987b; Kozak, 1991; Morris, 1995). It has been found difficult to estimate how
widespread uORFs are among the various organisms since only a minority of
databases containing precise and verified mapping of transcripts' 5'-ends. The
use of alternative transcription sites, alternative mRNA processing, and
alternative initiation codons also complicates the exact leader definition of a
given mRNA (see also Kozak, 2001a). In S. cerevisiae, however, a number of
uORF-comprising mRNAs has been positively identified (reviewed in McCarthy,
1998), and estimates suggest that up to 200 (out of approximately 6000) yeast
genes may contain this regulatory element (Vilela et al., 1998). In accordance
with published literature, throughout this work the term 5' untranslated regions
(5'-UTR) comprises the sequence of (regulative) uORFs although they are
technically translated; the expression ORF refers to a cistron encoding a full-
length gene product.
Although all uORFs can potentially undergo a full cycle of translation –
including initiation, peptide synthesis and termination, as shown by primer
extension experiments (Gaba et al., 2001) – there are differences in exactly
how they exert their restrictive influence on downstream initiation (Morris &
Geballe, 2000). The potential of a ribosome to reinitiate further downstream, as
well as the site at which it reinitiates can vary depending on both trans-acting
factors and the structure of the mRNA.
48
1. Introduction
1.4.2 uORF control in GCN4 translation: the stop codon context and eIF2 phosphorylation
In one of the most famous cases of mRNA-specific translational control, the
regulation of the GCN4 gene in yeast, amino acid starvation of cells leads to an
overall decrease in protein biosynthesis, while the expression of genes involved
in amino acid synthesis is up regulated. This is mediated via the translational
regulation of GCN4p, which itself presents a transcriptional activator
(Hinnebusch, 1984 and 1988). The initiation frequency at the GCN4 main ORF
is regulated by four uORFs that upon translation can exert a positive or negative
affect on reinitiation frequency. This control pathway is reviewed in detail by
Hinnbusch (1997).
In short, uORF1 and, to a lesser extent, uORF2 have been found to be
positive regulators of GCN4 translation since ribosomes terminating at these
ORFs are capable of downstream scanning and subsequent reinitiation. In turn,
uORF3 and especially uORF4 are promoting ribosome release at their
termination sites, thus preventing ribosomes to reach the downstream AUG of
the main ORF. The balance of initiations at the respective ORFs is regulated by
the availability of the active ternary complex Met-tRNAi-eIF2-GTP and the stop
codon context of the two upstream elements uORF1 and uORF4 that are most
relevant for regulation. The first uORF comprises an A/U-rich stop codon
context that is favouring reinitiation of translation whereas the G/C-rich
downstream element of uORF4 was shown to promote ribosome release (Grant
& Hinnebusch, 1994). These authors proposed that G/C-rich elements could
stall the terminating complex long enough for the subunits to dissociate,
probably by G/C base stacking or interaction of the mRNA site with tRNA, rRNA
or other parts of the mRNA. The GCN4 uORF4 10 nt downstream sequence
was also shown to act as a transferable element since it can reduce reinitiation
when placed downstream of the translationally non-restrictive YAP1 uORF
(Vilela et al., 1998). Sequences 5' of uORF1 have also been implied to be
required for efficient reinitiation, although the mechanism for this remains
unclear (Grant et al., 1994).
To (re)initiate translation, ribosomes need to be associated with factors
such as the active ternary complex Met-tRNAi-eIF2-GTP. In yeast, this is
regulated in response to environmental conditions via a kinase pathway. Under
starvation conditions, the kinase GCN2 is activated involving the binding of an
49
1. Introduction
uncharged tRNA and consequently phosphorylates the α subunit of eIF2
(reviewed in Hinnebusch, 1996). This enhances the affinity of eIF2 for the
nucleotide exchange factor eIF2B that is required to recycle the binary eIF2-
GDP complex (Yang & Hinnebusch, 1996). Since the exchange factor is
presumed to be considerably less abundant than eIF2, the phosphorylation of
20-30% of eIF2 is sufficient to effectively make eIF2B unavailable for nucleotide
exchange on eIF2-GDP complexes, resulting in a strong inhibition of translation
(Hershey, 1991; Rhoads, 1993).
By combining the two findings about stop codon context and eIF2
requirement, it was put forward that under normal growth conditions ribosomes
terminating at GCN4 uORF1 are able to resume scanning and acquire the
ternary complex in time to reinitiate at uORF4. Termination at this upstream
open reading frame then inhibits further scanning and translation of the GCN4
transcription factor. However, when eIF2 abundance is low, the scanning
ribosome supposedly takes longer to re-bind the ternary complex, thereby
'ignoring' the uORF4 start codon and initiating at the main ORF.
1.4.3 Reinitiation is affected by the intercistronic distance and the uORF length
In addition to intracellular eIF2 levels, the intercistronic distances between the
(u)ORFs plays a crucial role in determining whether the ribosomes can become
reinitiation-competent or not. Having terminated, ribosomes need a certain time
span to re-acquire the Met-tRNAi delivered by the ternary complex. This
minimum threshold is usually directly correlated with the length of the sequence
the ribosome can scan until it encounters a downstream AUG. Early information
came from Kozak (1987a) reporting that in human cells reinitiation becomes
more efficient when the distance between uORF and the next AUG was
lengthened without introduction of a significant secondary structure. An
intercistronic spacer of 79 nt entirely relieved the restriction on reinitiation
imposed by the uORF. In yeast cells, Grant and colleagues (1994) observed a
decrease in main ORF expression when the sequence between uORF1 and
GCN4 were shortened from 350 nt to 50 nt on a leader lacking the other three
uORFs. Similarly, under starvation conditions (and therefore lowered eIF2
levels) increasing the distance between uORF1 and uORF4 led to enhanced
ribosome release at uORF4 and therefore to a reduced frequency of reinitiation
50
1. Introduction
at the downstream GCN4 ORF (Abastado et al., 1991). In turn, shortening the
spacer from 200 nt to 32 nt resulted in increased GCN4 synthesis (Grant et al.,
1994). The requirement of a longer spacer for efficient reinitiation at the main
GCN4 ORF (>50 nt) in respect to the shorter intercistronic spacer needed for
reinitiation at uORF4 (32 nt) was explained by two types of model. The first
postulates that a slow initiation step (as at uORF4) would facilitate the binding
of the ternary complex whereas the second proposes that sequences at the
uORF4 start site can recruit additional, as yet uncharacterised, factors. Notably,
genetic experiments also implicate eIF3 in the Met-tRNAi rebinding step
(Garcia-Barrio et al., 1995). In all cases, the control would be kinetic. The
correlation between intercistronic distance and reinitiation competence has also
been observed in systems as diverse as HIV-1 (Luukkonen et al., 1995) and
fungi (Arst & Sheerins, 1996), for a review see Kozak (2002).
In analogy to the importance of the intercistronic spacer, the size of the 5'-
proximal ORF is thought to be a major constraint to reinitiation in eukaryotes.
This was initially deduced from studies in plant and animal viruses that produce
bicistronic mRNA in which the 3'-cistron is translationally silent (reviewed in
Kozak, 1978). While the scanning ribosome has to regain factors essential for
(re)initiation, especially the ternary complex, the elongating ribosome is thought
to lose them gradually. Ribosomes having translated only a short uORF
supposedly still retain these factors. Therefore, extending an upstream ORF
has a negative effect on reinitiation at a downstream ORF, but there are only
very few studies systematically exploring the uORF length – reinitiation
relationship. Luukkonnen et al. (1995) saw a gradual diminishment on viral
mRNA and predicted an almost complete absence of reinitiation capability when
uORFs reach a length of 55 codons (168 nt). Hwang and Su (1998) observed a
2-fold decrease in protein yield when uORF length was lengthened from 7 (24
nt) to 18 codons (57 nt) in a similar system. In rabbit reticulocyte lysate, Kozak
(2001) observed no change in reinitiation when the uORF was lengthened from
3 (12 nt) to 13 codons (42 nt) and then a gradual decrease. Protein yield of the
downstream ORF was decreased 3-fold when the uORF was expanded from 13
to 33 codons (102 nt), irrespective of the inserted sequence. Furthermore,
changing part of the uORF sequence to that of a pseudoknot reduced
reinitiation even more, most likely by slowing down the elongating ribosome.
51
1. Introduction
Kozak concludes that it is therefore difficult to determine a 'cut-off' length for
ORFs but that reinitiation competence is rather kinetically controlled, and that
slowing down of the ribosomal movement on the ORF therefore promotes the
release of initiation relevant factors.
1.4.4 Other factors governing reinitiation All upstream open reading frames and the main ORF of GCN4 were found to
recruit scanning ribosomes with about the same frequency (Grant et al., 1994).
However, in some cases the uORF initiation rate varies and therefore regulates
downstream reinitiation. Whether the initiation site is recognised or by-passed
by 'leaky scanning' can be affected by to the start codon context, the distance of
the start codon from the cap, the nature of the start codon (AUG or GUG), the
modulation by viral proteins and events such as ribosome shunting (see also
Section 1.2.4, Morris & Geballe, 2000).
Maybe surprisingly, also uORFs with initiation frequencies as low as 10%
have the potential to prevent ribosomes from reaching downstream ORFs by
causing ribosomes to stall either during elongation or during termination, thus
creating a blockade to subsequent scanning or translating ribosomes. In the
eukaryotic cases reported so far, the structure or sequence of the nascent
peptide chain is responsible for perturbing the normal events of uORF
translation, although the responsible peptides characterised so far do not share
a common consensus sequence (Morris & Geballe, 2000). For example, coding
sequence-dependent arrest of the ribosome at the uORF of the human
cytomegalovirus gpUL4 transcript was mapped to the termination codon by
primer extension experiments (Cao & Geballe, 1996). Similarly, the AdoMetDC
uORF failed to affect downstream initiation when the termination codon was
altered (Cao & Geballe, 1995; Mize et al., 1998). While in these two cases, the
nascent peptide inhibits peptidyl-tRNA hydrolysis during termination, the peptide
produced by the fungal uORFs of arg-2 and CPA-1 mRNAs most likely affects
the peptidyltransferase centre and thereby influences both terminating and
elongating ribosomes (Morris & Geballe, 2000).
Ribosome regulation by nascent peptides is also widespread in
prokaryotes and has been studied especially intensively in mRNAs encoding
antibiotics (see Lovett and Rogers, 1996, for review). For example, expression
of chloramphenicol acetyl transferase from the cat mRNA is subject to
52
1. Introduction
autoregulation conferred by ribosome stalling at an upstream leader. This
impediment is aided by low levels of the encoded antibiotics, which in turn
exposes the ribosome-binding site for translation of the main ORF. Similar to
the situation in eukaryotes, the peptides encoded by the inhibitory uORF are not
conserved, and they target the peptidyltransferase centre of the big ribosomal
subunit (Lovett and Rogers, 1996).
1.4.5 Reinitiation of translation in prokaryotes and eukaryotes The lack of reinitiation competence after translation of a full-length ORF also
explains the almost absolute absence of polycistronic transcripts in eukaryotes
(Kozak, 1989b). While prokaryotes can initiate translation at almost any site on
the mRNA via guidance of the Shine-Dalgarno sequence, the eukaryotic
translation apparatus is usually limited in its AUG recognition to the 5' 3'
scanning mechanism. This may explain the evolution of regulative mechanisms
in eukaryotes such as structured 5'-UTRs, leaky scanning, reinitiation and
ribosome shunt. Furthermore, prokaryotic ribosomes do not need to (re)acquire
initiation factors such as the ternary eIF2 complex. Therefore, reinitiation
competence in prokaryotes is not restricted by the length of the 5'-positioned
ORF. Furthermore, the situation for the influence of the intercistronic length is
reverse. Eukaryotic ribosomes regain the ability to initiate during
posttermination scanning, and backward scanning followed by reinitiation
upstream of the termination site is almost neglectable (Kozak, 2001b). In
contrast, 70S posttermination ribosomes seem to be able to shuffle back and
forth on the mRNA although only in a region obviously determined by the
ribosomes off-rate that lies on the order of one ribosome-equivalent length
(reviewed by McCarthy, 1998). Proximity of stop and start site enhances
reinitiation frequency as does the existence of a Shine-Dalgarno sequence. If
the intercistronic distance is too long, coupled reinitiation frequency declines but
ribosomes from the cellular pool can still enter directly at a downstream
translation initiation region (TIR). This is analogous to artificial eukaryotic
systems whereby a full-length 5'-ORF is followed by an internal ribosome entry
site (IRES) facilitating initiation at a downstream cistron (Kozak, 2001a).
In prokaryotes, the ribosome recycling factor RRF has been shown to
possess the property to release ribosomes from the mRNA, and its absence
resulted in highly elevated reinitiation of translation 3' to the termination codon
53
1. Introduction
(Janosi et al., 1998). In eukaryotes, no cytoplasmic RRF homologue has been
found (see Section 1.2.3), and the events that decide the fate of the
posttermination ribosome remain unclear. Although release factor eRF3 is a
strong candidate to possess the function of RRF in eukaryotes, the role of this
and other termination complex associated factors such as Pab1p, Upf1-3p have
to be elucidated before a model for the switch from ribosome recycling to
reinitiation can be proposed. Therefore, it also remains a distinct possibility that
posttermination ribosomes can remain on the mRNA following translation of a
full-length ORF and scan along the 3'-UTR to be channelled to the 5'-end. This
question will be addressed in part of this thesis.
1.4.6 uORFs can regulate transcript stability Beside their effect on the ability of (posttermination) ribosomes to (re)initiate,
uORFs can exert an influence on mRNA stability. Since their primary structure
is similar to mRNAs comprising premature termination codons (PTCs) they
have the potential to trigger accelerated mRNA degradation by the nonsense-
mediated pathway (NMD). However, the utilisation of this particular pathway to
reduce transcript abundance and gene expression has been reported for only a
restricted number of uORF-comprising mRNAs such as CPA1 (Ruiz-Echevarria
& Peltz, 2000) or aberrant transcripts like the his4-38 allele comprising a single
G insertion (Donahue et al., 1981; Leeds et al., 1991) and the inefficiently
spliced CYH2 precursor (He et al., 1993). Oliveira and McCarthy (1995)
reported that in yeast an uORF introduced by a single nucleotide mutation in an
artificial leader reduced the mRNA's steady-state levels to 20% accounting for
most of the simultaneously observed decrease in translational efficiency. Aided
by results from control constructs harbouring secondary structures, they
concluded that not translation inhibition per se but rather the mechanism by
which the recognition of the main ORF is rendered less efficient does determine
mRNA destabilisation. Indeed, the group could show later that the restriction of
posttermination scanning triggered accelerated mRNA decay (Linz et al., 1997).
An improved frequency of initiation and subsequent termination at the uORF
reducing downstream initiation was proposed to be responsible for the Upf1p-
dependent mRNA destabilisation. Notably, the artificial uORF tested preceded a
bacterial CAT ORF indicating that destabilisation does not require elements
unique to yeast transcripts.
54
1. Introduction
However, most of the natural uORF-containing mRNAs such as GCN4 and
(Ruiz-Echevarria et al., 1998) and YAP1 (Vilela et al., 1998) are not subject to
degradation via the NMD pathway. Therefore, Ruiz-Echevarria and Peltz (2000)
have proposed the existence of an 'escape mechanism' that relies on the
presence of an uORF-downstream stabiliser element (STE). These authors
showed that loosely defined, A/U-rich sequences confer transcript stability due
to their interaction with the RNA-binding protein Pub1p. The exact mechanism
how Pub1p-binding to STE signals the surveillance complex that the termination
is not premature and how the effects of the downstream sequence element
(DSE) responsible for triggering the NMD pathway are compensated remain
unclear. Furthermore, the not strictly defined A/U-rich sequences downstream
of GCN4 uORF1 have been shown to promote ribosome reinitiation, which in
turn could account for the observed increase in stabilisation regardless of the
Pub1p interaction (see also Section 1.4.2). The convenience of explaining NMD
by the occurrence of STEs and DSEs is also challenged by the finding that the
two uORFs of YAP2 attenuate gene expression at the level of mRNA turnover.
Described first by Vilela and colleagues in 1998 and then reinvestigated in
1999, the YAP2 5'-UTR causes accelerated mRNA decay that is largely
independent of the NMD factor Upf1p. No DSE- or STE-like element could be
identified in the YAP2 leader, but similarly to GCN4 uORF4 the destabilising two
uORFs were followed by a G/C-rich sequence promoting ribosome release and
preventing reinitiation. Thus, the authors describe a model, whereby uORF-
directed destabilisation is modulated by events at the termination site
determining whether ribosomes can resume scanning and reinitiate, or the
ribosomes are released from the mRNA triggering its decay. The ability for
ribosomes to reinitiate might also be the key to explain the absence of Upf-
dependent destabilisation by the GCN4 uORFs. Ruiz-Echevarria & Peltz (1996)
inserted a G/C-rich DSE downstream of uORF4 that triggered NMD. This effect
was relieved when the DSE was placed > 200 nt from the stop codon or an A/U-
rich STE was inserted between the termination site and the DSE. Obviously, the
decision at the translation termination site whether ribosomes are released or
proceed to reinitiate defines the fate of the mRNA and can be affected by
downstream sequences.
55
1. Introduction
Therefore, it remains to be tested whether the STE element exerts its
stabilising effect simply due to the sequence's A/U content and accordingly
facilitated reinitiation or because of interactions, e.g. with Pub1p (which in turn
could also promote reinitiation). The role of STEs is also somewhat complicated
by the proposal that there exists another class of these elements within ORFs
that requires translation to exert its positive effect (Peltz et al., 1993; Hagan et
al., 1995; Ruiz-Echevarria et al., 2001).
1.5 Objectives The recent progress in translational research has provided inspiring information
about the factors and the function of mRNA circularisation. The synergistic
enhancement in translation initiation was traced to the interaction of the proteins
associated with the transcripts termini; yet there has been no thorough
examination of the possibility that terminating ribosomes might be recycled
directly to the mRNA 5’-end. Since the major part of translation is performed by
ribosomes that have terminated at least once, this ‘channelling’ of subunits that
are protected from the recruitment by competing mRNAs might contribute
significantly to the overall translational efficiency of the individual transcripts.
Besides, very little is known about the fate of the posttermination ribosome
and whether the subunits dissociate immediately at the stop codon or if they
migrate into the 3’UTR, which has been shown to occur after translation of short
open reading frames. This study aims to fill this gap by blocking this putative
posttermination scanning step, and monitoring of translational efficiency and
mRNA stabilisation will reveal the contribution of this assumed intramolecular
recycling.
Another aspect of this work will focus on a methodical analysis of the
correlation of uORF length and ribosomal reinitiation competence by stepwise
truncation of a full-length ORF. This is expected to give evidence about the
differences of posttermination ribosome having translated either a short,
regulative uORF or a full-length cistron comprised by the majority of cellular
mRNAs. Additionally, some of the studies researching uORFs and reinitiation
suffer from the occurrence of leaky scanning along with reinitiation. Hence, one
of the objectives will be to rule out this mechanism obscuring the origin of
ribosomes initiating at the downstream ORF.
56
2. Materials and Methods
2. MATERIALS AND METHODS
2.1 Chemicals and enzymes Tables 2.1 and 2.2 provide an overview of the chemicals and enzymes used.
Throughout this section, all concentrations are in weight / volume (w/v) except
where stated differently. Table 2.3 gives an overview of RCF values and the
corresponding speed in rpm in common rotors. Chemical Supplier Remark agar Merck agarose Sigma RNA grade acetate BDH amino acids Sigma ammonium chloride BDH NH4Cl ampicillin Melford amp adenosine triphosphate
Sigma ATP
bacto-tryptone Difco bacto-peptone Difco bovine serum albumin Sigma BSA 5-bromo-4-chloro-3-indolyl phosphate disodium salt
Sigma BCIP
borate BDH calcium dichloride BDH CaCl2 ·
2H2O caesium chloride Sigma CsCl, 98% cap analogue NEBL m7GpppG creatine phosphate Sigma cycloheximide Sigma D-cuciferin Sigma diethyl pyrocarbonate Sigma DEPC dimethyl formamide BDH DMF dithiothreitol Promega DTT ethylene diamine-tetraacetic acid
Sigma EDTA
ethanol BDH ethidium bromide Sigma EtBr ficoll Sigma ficoll 400 formaldehyde Fisher 37% formamide Gibco
BRL deionised
32P γ-ATP ICN 7500 mCi/ml
glass beads BDH 0.4 mm galactose BDH GAL glucose BDH GLU glycerol Merck glycine BDH glycylglycine Sigma guanosine triphosphate
Sigma GTP
Chemical Supplier Remark isopropanol BDH kanamycin Melford kan lithium acetate Sigma LiAc mannitol BDH magnesium acetate BDH MgAc magnesiumoxid acetate
BDH MgOAc
magnesium chloride BDH MgCl2· 6H2O
magnesium sulfate BDH MgSO4 methanol BDH 3-(N-morpholino) propanesulfonic acid
BDH MOPS
nitro blue tetrazolium Melford NBT phenylmethyl-sulphonyl fluoride
BDH PMSF
polyacrylamid solution
Severn Biotech
30% or 40%
polyethylene glycol BDH PEG4000, MW 4000
polyvinyl-pyrollidone BDH PVP potassium acetate BDH Kac potassium hydroxid BDH KOH potassiumoxid acetate BDH KOAc salmon sperm DNA Sigma preparation
according to Sambrock et al. (1989)
sodium acetate BDH NaAc sodium chloride BDH NaCl sodium dodecyl sulfate
BDH SDS
sodium bicarbonate BDH NaHCO3 sodium phosphate BDH NaH2PO4 sodium citrate BDH NaCitrate bis(2-hydroxyeth yl)-imino-tris(hydro xymethyl)methane
BDH Tris
urea BDH yeast extract Merck yeast nitrogen base Difco YNB (with
ammonium)
Table 2.1. Chemicals used. Abbreviations are mentioned in the remark column.
57
2. Materials and Methods
Enymes & antibodies Supplier Remark AMV Roche avian myeloblastosis virus reverse transcriptase anti-FLAG, from rabbit Santa Cruz
Biotechnology dilution 1/1000
anti-rabbit, from goat Sigma dilution 1/10000, conjugate with alkaline phosphates (AP)
DNase Promega Klenow fragment Roche PNK NEBL polynucleotide kinase restriction enzymes NEBL, Roche various RNase A Promega Freed of DNases-according to Sambrook et al.
(1989) RNase H Promega RNasin Promega T7 RNA Polymerase NEBL
Table 2.2. Enzymes and antibodies.
Max. RCF (x g) rpm Centrifuge, rotor 860 3,000 Heraeus biofuge pico 1,240 3,000 Beckman JA-17 2,200 4,000 Beckman JA-17 2,400 5,000 Heraeus biofuge pico 8,800 8,000 Beckman JA-17 16,100 13,000 Howe Sigma 1K13 35,000 30,000 Beckman JA-25.50 230,000 50,000 Beckman 70.1 Ti 270,000 39,000 Beckman SW40 Ti
Table 2.3. Correlation of rpm and RCF values in centrifuges and rotors used. RCF, relative centrifugal force; rpm, revolutions per minute
2.2 Cell methods
2.2.1 Strains and cell culturing
Saccharomyces cerevisiae strains
W303 [MATa/MATα ade2-1/ade2-1 his3-11/his3-11 leu2-3,112/leu2-3,112
trp1∆1/ trp1∆1 ura3-52/ura3-52 can1-100/can1-100], see Thomas & Rothstein
(1989)
NT33-5 [relevant genotype: ura3-52 leu2-3,112 cca1-1] as described in Wolfe et
al. (1996)
YAS1947/pTIF4631-213TRP1CEN This strain was derived from YAS1947
[MATα ade2 his3 leu2 trp1 ura3 tif4631::LEU2 tif4632::ura3
pTIF4632URA3CEN, Tarun et al. (1997)] by plasmid shuffling. For this, the
original strain containing the URA3 plasmid was transformed with the TRP1
marker containing pTIF4631-213 vector (Tarun et al., 1997). Positive
58
2. Materials and Methods
transformants were screened in several rounds of liquid culturing, streaking and
spreading onto YNB ura ade his or YNB trp ade his plates (see below) for loss
of the URA3 marker.
Yeast culturing
For the experiments, the yeast strains were grown at 30°C (NT33-5 at 25°C or
37°C) in YNB media (yeast nitrogen base, 6.7 g/l) and glucose or galactose (20
g/l) prepared in accordance with Sherman et al. (1986). Supplementation with
amino acids was to complement auxotrophies and to select for the respective
plasmid markers. Final concentrations [in mg/l] were: ade [40], ura [20], trp [20],
his [20], arg [20], tyr [30], leu [40], lys [30], ile [30], phe [50], val [150], thr [200],
ser [400].
By varying induction time and the carbon source, optimal conditions for
IRP1 expression were found to be as follows: 20 ml pre-culture was grown to
log-phase in YNB glucose, centrifuged for 5 minutes at 3500 rpm and washed
twice with sterile water. The cells were then resuspended and grown in YNB
galactose (YNB glucose for the control) to an OD600 of 0.8-0.9.
Escherichia coli strain and culturing
The E. coli strain Top-10F' [F'{lacIq Tn10(Tetr)} mcrA ∆(mrr-hsdRMS-mrcBC)
F80lacZ∆M15 ∆lac74 recA1 deoR araD139 ∆(ara-leu)7697 galU galK rpsL
endA1 nupG, obtained from Invitrogen] was used for DNA amplification. E. coli
media LB was prepared as described in Sambrook et al. (1989) to a final
concentration of 1% bacto-tryptone, 1% NaCl and 0.5% yeast-extract.
2.2.2 Escherichia coli transformation E. coli was transformed according to the CaCl2 method (Huff et al., 1990). For
this, bacterial cells were grown over night at 37°C in 5 ml of LB medium with
shaking (200 rpm). 2 ml of this culture were added to 100 ml LB medium, and
the cells were allowed to grow to an OD600 of 0.6. Subsequently, the cells were
cooled on ice for 10 minutes and then harvested by centrifugation for 10
minutes at 4°C and 2,200 x g. The cell pellet was resuspended in autoclaved 50
ml ice-cold 0.1 M CaCl2 and cooled on ice for 30 minutes. Afterwards, the
bacteria were pelleted again for 10 minutes at 4°C and 2,200 x g, resuspended
in 4 ml ice-cold 0.1 M CaCl2 and kept on ice over night. Sterile glycerol was
59
2. Materials and Methods
added to a final concentration of 20%, and aliquots of the competent cells were
snap frozen in liquid nitrogen and stored for later use at -80°C.
For transformation, 100 µl of competent cells were mixed with 50 ng
plasmid DNA and incubated on ice for 30 minutes. The cells were heat-shocked
for 50 seconds at 42°C, cooled on ice for 2 minutes and then incubated with
900 µl LB medium at 37°C with shaking (200 rpm) for 45 minutes. Next, the
cells were collected at 2,400 x g and spread on LB plates (LB with 1.5% agar).
The plates contained 100 µg/ml of the appropriate antibiotic (ampicillin or
kanamycin) selecting thereby for successfully transformed cells carrying the
plasmid with the corresponding resistance gene. Colonies were formed when
the plates were incubated over night at 37°C, short-term storage was at 4°C
thereafter.
2.2.3 Saccharomyces cerevisiae transformation Yeast transformation was performed according to the lithium acetate method
(Schiestl & Gietzl, 1989). Cells were inoculated in 20 ml YEPD (10 g/l yeast
extract, 20 g/l bacto-pepton and 3 g/l glucose) and incubated with shaking (180
rpm) over night at 30°C. On the next day, about 1 ml of the overnight culture
was added to 20 ml fresh YEPD to give 5x106 cells/ml (OD600 ~ 0.25). The
culture was allowed to grow to a density of 2x107 cells/ml (OD600 ~ 0.8) and
then harvested by centrifugation at 1,240 x g.
The cell pellet was washed twice by resuspending in 20 ml sterile water
and centrifuging as before. Subsequently, the cells were transferred to a 2 ml
micro tube and washed two times in 1 ml freshly prepared TELiAc buffer (10
mM Tris pH 7.7, 1 mM EDTA, 100 mM LiAc), collection was at 860 x g. Finally,
the cells were resuspended in 1 ml TELiAC to give 2x109 cells/ml.
For transformation, 5 µg of each plasmid DNA and 20 µg salmon sperm
DNA were incubated with 50 µl (108) freshly prepared yeast cells and 300 µl
PEG/LiAc (50% PEG4000, 10 mM Tris pH 7.7, 1 mM EDTA, 100 mM LiAc) at
30°C for 30 minutes. After a 10 minutes (2 minutes for heat sensitive strains)
heat-shock at 42°C, the cells were collected by centrifugation at 860 x g for 5
minutes. Resuspended in 100 µl sterile water and plated onto appropriate
selective YNB-plates (YNB with 1.5% agar and 2% glucose added), yeast
colonies appeared 2-3 days later when incubated at 30°C (or 25°C for heat
sensitive strains).
60
2. Materials and Methods
2.3 Recombinant DNA methods
2.3.1 DNA manipulation DNA cloning was performed using standard methods such as PCR, DNA
restriction and ligation, (Sambrook et al., 1989). To obtain the RT-PCR product
of actin and the spacer – IRE sequences (see below), the TA cloning kit
(Invitrogen) was employed following manufacturer's instructions. Mutations were
introduced by site-directed mutagenesis via PCR or insertion of double stranded
oligodeoxyribonucleotides. All constructs were confirmed by fluorescence
sequencing (Sequencing service, UMIST).
2.3.2 Screening of E. coli transformants Screening to detect transformants with plasmids carrying the desired DNA
sequence was performed by standard methods (Sambrook et al., 1989). For
mini-preparation of DNA by alkaline hydrolysis and subsequent restriction
analysis, single E. coli transformant colonies were inoculated over night at 37°C
in 2 ml LB medium with shaking (200 rpm). On the next day, 1.5 ml of this
culture was collected for 5 minutes at 2,400 x g and resuspended in 200 µl
solution I (50 mM glucose, 2 mM Tris.Cl pH 8.0, 10 mM EDTA pH 8.0, 100
µg/ml RNase A). Lysis of the cells was by addition of 400 µl solution II (1%
SDS, 0.2 M NaOH) and incubation for 2 minutes on ice. Mixing with 200 µl
solution III (3 M KAc, 5 M acetate) precipitated cell debris and genomic DNA.
After 5 minutes incubation on ice, the plasmid DNA was separated from the cell
pellet by centrifugation at 4°C and 16,100 x g for 5 minutes. The supernatant
was precipitated by adding 2 volumes of isopropanol. Having washed the pellet
with 70% ethanol, the dried DNA was resuspended in an appropriate volume to
give a concentration of about 1 µg/µl. The vector was subject to restriction
analysis to screen for recombinant DNA, positive samples were confirmed by
fluorescence sequencing.
Alternatively, up to 25 colonies were resuspended in 20 µl of a standard
PCR mix and subjected to amplification with plasmid-specific primers. After
agarose gel analysis, the plasmids of positive candidates were mini-preped
using a nucleo-spin kit (Macherey-Nagel) and sequenced.
61
2. Materials and Methods
2.3.3 CsCl discontinuous gradient DNA purification Conventional DNA preparation implies treatment with RNases that can result in
mRNA degradation and in consecutive aberrant in vitro transcription reactions.
Hence, template plasmids were purified in CsCl gradients (adapted from
Sambrook et al., 1989). For this, DNA was prepared according to the mini-prep
protocol in Section 2.3.2 but without RNase treatment and resuspended in a
solution of 6.82 g CsCl and 3.91 g TE (10 mM Tris.Cl, 1 mM EDTA pH 8.0). To
this 0.64 g/g CsCl solution, ethidium bromide was added to a final concentration
of 0.43 mg/ml. The resulting solution was mixed and centrifuged for 5 minutes
at 8,800 x g. The clear red supernatant was loaded onto 6.5 ml of 0.75 g/l CsCl,
prelayed in a 13 ml Beckman Quickseal tube. After sealing of the tube,
centrifugation was at 15°C and 230,000 x g over night. Finally, the (lower) DNA
band was recovered with a syringe, the ethidium bromide separated by 6
extractions with butan-1-ol in TE and the remaining supernatant precipitated
with 2% polyethylene glycol. The pure DNA pellet was washed with 70%
ethanol and resuspended in water.
2.3.4 Plasmid constructs All oligodeoxyribonucleotides used in this research were obtained from Sigma-
Genosys or MWG Biotech and are listed in Table 2.4.
The IRP1-IRE system and poly(G)-containing constructs
Iron-response-element binding protein IRP1 (Butt et al., 1996; Gray et al., 1993)
was cloned as a 2.9 kb NdeI / XbaI fragment from YCpSUP-IRF (Oliveira et al.,
1993a) into YCp33Supex2 (Supex2, Oliveira et al., 1993b). Expression from this
plasmid is regulated by the galactose-inducible Gal-PGK fusion (GPF) promoter
(Vega Laso et al., 1993).
Plasmid FL comprising the constitutive promoter TEF1 and the firefly
luciferase ORF followed by a PGK1-derived 3'-UTR corresponds to YCp22FL1
in Oliveira et al. (1993a). FL-5'IRE with the IRE sequenced placed 9 nt into the
5'-UTR equals construct IRE-wt in Oliveira et al. (1993b), see also Table 2.5. To
create FL-derived constructs with the IRE in the 3'-UTR, synthetic DNA was
inserted between the XbaI and EcoRI sites of FL. PCR products derived from
reactions with the respective positive-strand primer (XbaIIRE(+), XbaI15IRE(+)
and XbaI30IRE(+)) and EcoRIIRE were used to achieve constructs FL-15-IRE,
62
2. Materials and Methods
FL-24-IRE and FL-39-IRE. Likewise, annealed XbaI-poly(G)+/- primers were
ligated into the XbaI site of FL to produce FL-6-pG (Table 2.5). Primer Sequence) 5’ATG mut GGAATTCCATATGGATTACAAGGACGACGACGACAAGACTAAGGATTC 5’TEF CGGTCTTCAATTTCTCAAGTTTCAG 3’ATG mut GTAATCTAGAATTCTACATTAGTCACCGGCAAAACCGGCTTTACACAAACCAGAACC actDEL105- CGATAGATGGGAAGTCTAGAATTCTACATTAGTCACCGGCAAAACCGGC ActStop30+ GCTTTGGTTATTTAATGTAGAATTCGTATGTGTAAAGCC ActStop30- GGCTTTACACATACGAATTCTACATTAAATAACCAAAGC ActStop298+ GCCCCAGAATAATGTAGAATTCTTTTGACTGAAGCTCC ActStop298- GGAGCTTCAGTCAAAAGAATTCTACATTATTCTGGGGC ActStop640+ CGTGACATCAAGTAATGTAGAATTCACGTCGCCTTGGACTTCG ActStop640- CGAAGTCCAAGGCGACGTGAATTCTACATTACTTGATGTCACG ActStop931+ CCAGGTATTGCCTAATGTAGAATTCAGGAAATCACCGCTTTGGC ActStop931- GCCAAAGCGGTGATTTCCTGAATTCTACATTAGGCAATACCTGG ATGATmut+ GGAATTCCATATGATGTACAAGGACGAC ATGcorr- CAAAATATAATCGGTTCTAGAATTCTACTTTA ATGmut30- GGAATTCCATATGATGGATTACAAGGACGAC ATGmut60- GTAATCTAGAATTCTACATTAAATAACCAAAGCAGCAACCTCAG EcoRIIRE CCGGAATTCTTCTCGAGTTTTGTGTTTCTTAAGTTCAAGC EcoRIT(20) CCGGAATTCTTTTTTTTTTTTTTTTTTTT FlagATGdel+ GTTGTTTCTTAACATTTGGATTACAAG FlagATGdel- CTTGTAATCCAAATGTTAAGAAACAAC FLAGdel12- CCTCAGAATCCATATCTAGAATTCTACATTACTTGTAATCC FLAGdel30- CAATAACCAAAGCATCTAGAATTCTACATTAAGACTTGTCATCG IREXho+ CCGCTCGAGAATTATCTACTTAAGCTTC LUC-1Pro (+) TCGAGCAAACCGTAAT LUC-1Pro (-) CTAGATTACGGTTTGC LUC-2Pro (+) TCGAGCCCGTTGTAAT LUC-2Pro (-) CTAGATTACAACGGGC LUC10ds (+) TCGAGCAAATTGTAAGCGGTTACCTT LUC10ds (-) CTAGAAGGTAACCGCTTACAATTTGC LUC-1,2pro10ds (+) TCGAGCCCGCCGTAAGCGGTTACCTT LUC-1,2pro10ds (-) CTAGAAGGTAACCGCTTACGGCGGGC LucRev1546 CTCCTCCGCGCAACTTTTTCGCGGTTG LUCstopstop (+) TCGAGCAAATTGTAATAAT LUCstopstop (-) CTAGATTATTACAATTTGC LucTerm+ GAAGGGCGGAAAGTCCAAATTG LUCXho (+) GAAGGGCGGCTCGAGCAAATTG LUCXho (-) CAATTTGCTCGAGCCGCCCTTC Nde-Actin+ GGGAATTCCATATGGATTCTGAGGTTGCTGC NdeFlag+ TATGGATTACAAGGACGACGATGACAAGAC NdeFlag- TAGTCTTGTCATCGTCGTCCTTGTAATCCA P3XM+ CTATTATTTATCTCGAGATGATTATTAAG P3XM-New CTTAATAATCATCTCGAGATAAATAATAG PGK1TERM-new CCCAAGCTTTAACGTTCGCAGAATTTTCG PGK3UTRRev58 GCGTAAAGGATGGGGAAAGAGAAAAG PHS1-F GCACCGCCGCCGCAAGGAATGG RP131 GAAGTCAACTCTGATGAAGAC uORF1ds50+ TATGTCTAGAACCGATTATATTTTGTTTTTAAAGTAGATCTTCTCGAGAAAAAC uORF1ds50- TAGTTTTTCTCGAGAAGATCTACTTTAAAAACAAAATATAATCGGTTCTAGACA XbaI-Actin- CTAGTCTAGATTAGAAACACTTGTGGTGAACG XbaI-poly(G)+ CTAGAGGGGGGGGGGGGGGGGGGT XbaI-poly(G)- CTAGACCCCCCCCCCCCCCCCCCT XbaI15IRE(+) GCTCTAGAATTTATAAAAATTATCTACTTAAGCTTC XbaI30IRE(+) GCTCTAGAATTTATAAAAACAATTACCACAAAAATTATCTACTTAAGCTTC XbaIIIRE(+) GCTCTAGAAATTATCTACTTAAGCTTC XhoI-p(G)18+ TCGAGGGGGGGGGGGGGGGGGGC XhoI-p(G)18- TCGAGCCCCCCCCCCCCCCCCCC
Table 2.4. Sequences of oligodeoxyribonucleotides. Sequences of all primers used in this study are given in the 5' 3' orientation.
63
2. Materials and Methods
For insertion of IRE close to the poly(A) site, first a XhoI-free reference
construct was created by replacing the 5'-UTR of FL BamHI / NdeI with the
longer uORF-free YAP1 leader of plasmid p∆uY1 (Vilela et al., 1998). Then,
with primers P3XM+ and P3XM-New, a new XhoI site was introduced 141
nucleotides into the 3'-UTR. The resulting construct FL' (see also Table 2.5)
displayed no significant difference to FL with respect to in vivo luciferase
expression and LUC mRNA stability (data not shown). To obtain plasmid FL-
141-IRE, the IRE sequence (derived from a XhoI restricted PCR reaction with
primers IREXho+ and EcoRIIRE) was inserted as a duplicate into the XhoI site
of FL'.
A 5'-UTR sequences FL AATTATCTACTTAAGAACACAAAACTCGAGAACATATG
FL' GGATCCTTACCGATTAAGCACAGTACCTTTACGTTATATATAGGATTGGTGTTTAGCTTTTTTTCCTGAGCCCCTGGTTGACTTGTGCAAGAACACGAGCCATTTTTAGTTTGTTTAAGGGAAGTTTTTTGCCACCCAAAACGTTTAAAGAAGGAAAAGTTGTTTCTTAACATATG
FL-5'IRE AATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAACACAAAACTCGAGAACATATG
B 3'-UTR sequences FL TAATCTAGAATTC – 143 nt – p(A)
FL-15-IRE TAATCTAGAAATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAAACACAAAACTCGAGAAGAATTC – 143 nt – p(A)
FL-24-IRE TAATCTAGAATTTATAAAAATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAAACACAAAACTCGAGAAGAATTC – 143 nt – p(A)
FL-39-IRE TAATCTAGAATTTATAAAAACAATTACCACAAAAATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAAACAAACTCGAGAAGAATTC –143 nt–CAA p(A)
FL-141-IRE TAATCTAGAATTC – 116 nt – CTCGAGAATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAAACACAAAACTCGAGAATTATCTACTTAAGCTTCAACAGTGCTTGAACTTAAGAAACACAAAACTCGAG – 21 nt – p(A)
FL-6-pG TAATCTAGAGGGGGGGGGGGGGGGGGGTCTAGAATTC – 143 nt – p(A)
FL-131-pG TAATCTAGAATTC – 116 nt – CTCGAGGGGGGGGGGGGGGGGGGCTCGAG – 21 nt - p(A)
C sequences of actin ORF truncations and intercistronic spacer ACTd-LUC d15 d33
ATG GAT TAC AAG GAC GAC GAT GAC AAG ACT ATG GAT TCT GAG GTT GCT GCT TTG GTTd63 d105 ______inter-ATT GAT AAC GGT TCT GGT ATG TGT AAA GCC GGT TTT GCC GGT GAC TAA TGTAGAATTCT________________cistronic_spacer________________ LUC AGAACCGATTATATTTTGTTTTTAAAGTAGATCTTCTCGAGAAAAACT ATG
ACTd ∆iAUG-LUC
ATG GAT TAC AAG GAC GAC GAC GAC AAG ACT AAG GAT TCT GAG GTT GCT GCT TTG GTT ATT GAT AAC GGT TCT GGT TTG TGT AAA GCC GGT TTT GCC GGT GAC TAA AGTAGAATTCTAGAACCGATTATATTTTGTTTTTAAAGTAGATCTTCTCGAGAAAAACT ATG
AUGAUG ACTd-LUC
ATG ATG TAC AAG GAC GAC GAC GAC AAG ACT AAG GAT TCT GAG GTT GCT GCT TTG GTT ATT GAT AAC GGT TCT GGT TTG TGT AAA GCC GGT TTT GCC GGT GAC TAA AGTAGAATTCTAGAACCGATTATATTTTGTTTTTAAAGTAGATCTTCTCGAGAAAAACT ATG
∆AUG ACTd-LUC
TTG GAT TAC AAG GAC GAC GAC GAC AAG ACT AAG GAT TCT GAG GTT GCT GCT TTG GTT ATT GAT AAC GGT TCT GGT TTG TGT AAA GCC GGT TTT GCC GGT GAC TAA AGTAGAATTCTAGAACCGATTATATTTTGTTTTTAAAGTAGATCTTCTCGAGAAAAACT ATG
Table 2.5. Sequences of FL-derived UTRs and ATG-mutated truncated actin-luciferase constructs. Sequences are given in the 5' 3' orientation. (A) 5'-UTR sequences (starting with the first transcribed nucleotide) of FL, FL' and FL-5'IRE and (B) 3'-UTR sequences of FL and 3'-UTR modified FL constructs. The bold ATG and TAA triplets refer to the luciferase start and stop-codon, respectively. The poly-guanidine stretch pG and the stems of the IRE hairpin-loop are underlined; p(A) specifies the polyadenylation site. (C) Truncated actin ORF and intercistronic sequence of actin-luciferase constructs. ATGs are indicated in bold, mutations relative to the preceding construct are underlined. d15, d33 d63 and d105 refer to the last codon of constructs with uORFs truncated to 15, 33, 63 and 105 nt, respectively. LUC specifies the luciferase start codon.
64
2. Materials and Methods
Similarly, synthetic DNA (annealed oligodeoxyribonucleotides XhoI-p(G)18+
and XhoI-p(G)18-) was inserted into the XhoI site of FL' creating plasmid FL-
131-pG (Table 2.5).
For the experiments in strain YAS1947, plasmid BEFL was established to
place the LUC expression cassette of FL on a plasmid with the yeast ADE2
marker. For this, FL was cut at the sites PstI and HindIII, and the resulting DNA
fragment was (partly blunt-end-) ligated into BEVY-A (Miller 3rd et al., 1998) cut
at PstI and EcoRI. Construct BEFL-5'IRE was generated by replacing the
BamHI – NdeI fragment with the 5'-UTR of FL-5'IRE. BEFL-39-IRE is the result
of substituting the BEFL EcoRI – EcoRI section with the corresponding
fragment of FL-39-IRE.
LUC stop codon context modifications
FL* was derived from FL by replacing the 5'-UTR with the one of FL' and
introducing an XhoI site upstream of the LUC stop codon (Figure 3.5A) using a
two step PCR approach with primer pairs 5'TEF / LUCXho (-) and LUCXho (+) /
PGKTERM-new. This facilitated the change of the stop codon sequence by
insertion of deoxyribonucleotide pairs LUC-1Pro, LUC-2Pro, LUC-12Pro10ds,
LUC10ds and LUCstopstop as described in Figure 3.5A. FL* showed no
significant difference to FL regarding in vivo luciferase expression and LUC
mRNA stability (data not shown).
ACT1 cloning and LUC cassette appendage
The S. cerevisiae actin gene ACT1 was amplified from W303 cell lysates by RT-
PCR as described in RNA methods (Section 2.4.3) with primers Nde-Actin+ and
XbaI-Actin- introducing the conservative mutation A108C. The resulting DNA
was cloned NdeI / XbaI in vector FL' and consequently FLAG-tagged by
insertion of synthetic DNA (annealed primers NdeFlag+ / NdeFlag-) into the
NdeI site, mutating thereby the introduced second NdeI site. ACT-LUC and all
other bicistronic luciferase constructs were derived from the respective
monocistronic FLAG-tagged ACT construct by insertion of an XbaI / XbaI
spacer-luciferase cassette. This element was established by ligation of the
annealed primers uORF1ds50+/- into plasmid FL's NdeI site and comprises the
LUC ORF preceded by a 59 nucleotide intercistronic spacer sequence derived
from the reinitiation competent GCN4 uORF1 downstream sequence (Table
65
2. Materials and Methods
2.5). Constructs ACT-pG-LUC and ACT-119-LUC were established by insertion
of a 18-guanosine stretch into the XhoI-site of ACT-LUC or by duplication of the
intercistronic spacer.
Premature termination codon constructs – series ACTptc and ACTptc-LUC
For insertion of premature stop codons in ACT1, the FLAG-actin ORF was
mutated by a two-step PCR downstream of position 960, 699, 324 or 60 to 5'-
TAATGTAGAATTC-3' using outside primers 5'Tef and PGKTERM-new and
internal primers ActStop30+/-, ActStop298+/-, ActStop640+/- and ActStop931+/-
. Thereby, the stop codon UAA and an invariable 10 nucleotides downstream
sequence was introduced giving rise to the series ACTptc-LUC.
C-terminal truncation of actin ORF – series ACTd and ACTd-LUC
In the truncated constructs, the 3'-end of the actin ORF was deleted either by
religation of the PTC-containing plasmids after restriction with EcoRI or by
deletion of sequences 3' of position 105, 33 and 15 via PCR with primers
actDEL105-, FLAGdel30- and FLAGdel12- (Table 2.5).
Mutation of actin AUGs – series ACTd∆iAUG-LUC, AUGAUGACTd-LUC and
ACTd∆iAUG-LUC
In order to eliminate internal AUGs, ACT-LUC constructs with an actin ORF
truncated to 105, 63, 33 or 15 nucleotides were mutated by PCR as shown in
Table 2.5 giving thereby rise to the ACTd∆iAUG-LUC series. Primers employed
were 5'ATG mut, 3'ATG mut, ATGmut30-, ATGmut60- and ATGcorr-.
In the AUGAUGACTd-LUC series, another PCR mutation with primer
ATGATmut+ changed the second actin codon of ACTd105∆iAUG-LUC,
ACTd63∆iAUG-LUC and ACTd33∆iAUG-LUC to AUG, introducing a tandem
start codon. Similarly, in a two-step PCR (primers 5'TEF / FlagATGdel+ and
PGKTERM-new / FlagATGdel-), the actin start codon was mutated to UUG in
the ∆AUGACT-LUC constructs, leaving the LUC start codon as the first AUG on
the mRNA (Table 2.5).
For experiments in strain NT33-5, the ACTd∆iAUG-LUC constructs were
subcloned NdeI / HindIII in the URA3 vector p∆uY1 (termed FL", Vilela et al.,
1998) carrying a luciferase expression cassette identical to the one of FL'.
66
2. Materials and Methods
Vectors for in vitro transcription
Vectors pHST7-FL and pHST7-FL-39-IRE were derived from pHST7 (Koloteva
et al., 1997) by exchanging the NdeI – XbaI fragment with plasmids FL and FL-
39-IRE, respectively. This also removed an aberrant 3'-UTR contained in the
original plasmid pHST7-5'IRE as described in Koloteva et al. (1997).
2.4 RNA methods Glassware, plasticware and solutions used in the procedures described in this
section were treated with diethyl pyrocarbonate (DEPC) and autoclaved where
appropriate to prevent contamination with RNases.
2.4.1 Total yeast RNA extraction Total RNA was isolated using a modified version of the hot phenol procedure
(Rose et al., 1990; Köhrer & Domdey, 1991). In detail, 20 ml of each yeast
culture at OD600 ~ 0.8 were harvested for 5 minutes at 1,240 x g, washed once
with 20 ml ice-cold water and finally resuspended in 500 µl AE buffer (50 mM
sodium acetate, 10 mM EDTA pH 5.3) and 100 µl 10% SDS. RNA was
extracted in two cycles of hot phenol extraction (5 minutes shaking at 65°C,
snap-freezing in liquid nitrogen and 5 minutes centrifugation at 16,100 x g) and
consecutive two-fold phenol:chloroform:isopropanol (25:24:1, v/v/v) extraction
(5 minutes vortexing, 5 minutes centrifugation at 16,100 x g). Precipitation of the
aqueous phase was with 0.3 mM sodium acetate and ethanol in a final volume
of 2 ml. Samples were stored in ethanol short-term at -20°C or long-term at
-80C.
Before use, the samples were centrifuged at 4°C and 16,100 x g for 10
minutes, washed with 70% ice-cold ethanol and centrifuged as before. Then,
the dried pellet was resuspended in 20-50 µl DEPC-treated water by 3-5 cycles
of vigorous vortexing (1 minute) and cooling (2 minutes on ice).
2.4.2 Northern blot
Formaldehyde agarose gel electrophoresis and blotting
20 µg of total yeast RNA were denatured for 15 minutes at 65°C in a RNA
denaturing mix (6% formaldehyde, 50% formamide, 1x MOPS buffer [200 mM
MOPS pH 7.0, 50 mM sodium acetate, 10 mM EDTA pH 8.0]). Denaturation
was stopped by cooling on ice and addition of formamide loading buffer (1
67
2. Materials and Methods
mg/ml bromophenol blue, 1 mg/ml xylene cyanol FF in 0.1 mM EDTA pH 8.0
and 100% formamide). The samples were separated on a 1.3% formaldehyde
agarose gel (1.3% agarose, 1x MOPS, 2.24 M formaldehyde in DEPC-treated
water) with continuous mixing of the MOPS buffer according to Sambrook et al.
(1989).
The RNA was then blotted onto nylon membranes (Hybond-N, Amersham
Pharmacia Biotech) according to manufacturer's instruction. The following day,
the RNA was cross-linked by UV-radiation (1,200 units in a Stratagene UV
Stratalinker 2400) and baked to the membrane at 80°C for 2 hours.
Hybridisation with γ-labelled primers 32P γ-labelling of 25 pmol deoxyribonucleotides was with polynucleotide kinase
following manufacturer's instruction. The end-labelled primer was purified from
unincorporated 32P γ-ATP using Sephadex G-25 columns (NAP-5, Amersham
Pharmacia Biotech). With respect to the sequence to be detected, the labelled
primer(s) – RP131 (PGK1 recognition), LucRev1546 (LUC), NdeFlag- (FLAG)
or PGK3UTRrev58 (FL 3'-UTR) – were denatured together with 250 µg salmon
sperm DNA. Following pre-hybridisation for at least 3 hours in 50 ml,
hybridisation was at 50°C over night in 10 ml fresh (pre-) hybridisation solution
according to Kuhn et al., 2001 (5X SSC [20x: 3 M NaCl, 300 mM NaCitrate], 20
mM NaH2PO4 pH 7.2, 7% SDS, 1x Denhardt solution [50x: 1% BSA, 1% ficoll,
1% polyvinyl-pyrollidone]). The membranes were then washed according to
Kuhn et al. (2001) in Wash Solution 1 (3x SSC, 10x Denhardt solution, 5%
SDS, 25 mM NaH2PO4 pH 7.2) and Wash Solution 2 (1x SSC, 1% SDS) at
50°C. If necessary, the membranes were stripped in 0.1x SSPE pH 7.4 (20x: 3
M NaCl, 200 mM NaH2PO4, 20 mM EDTA), 0.1% SDS at 80°C for subsequent
rehybridisation. Results were quantified on a Molecular Dynamics Typhoon
8600 PhosphorImager using the ImageQuant software, version 5.1.
2.4.3 RT-PCR 1 µg of total yeast mRNA, isolated from cell extracts as described in 2.4.1 was
subject to a first strand cDNA synthesis reaction with AMV reverse
transcriptase. RNA and 25 pmol of poly(A)-tail specific primer EcoRI-T(20) were
denatured at 65°C for 5 minutes and then cooled down to 30°C. The reaction
was started by the addition of the remaining components (according to manual
68
2. Materials and Methods
from Roche) to a final volume of 20 µl and incubation at 42°C. The reaction was
stopped after 20 minutes by cooling the mix on ice. 4 µl of the reaction mix were
used to amplify the luciferase 3'-UTR in a subsequent PCR with primers EcoRI-
T(20) and LucTerm+. The obtained PCR product was sequenced directly with
LucTerm+.
2.4.4 poly(A) length determination by RNase H treatment For the poly(A)-tail length determination, 25 µg of total yeast RNA extract were
incubated with RNase H (Promega) and either a combination of
oligodeoxyribonucleotides LucRev1542 and EcoRI-T(20) or LucRev1542 alone
(25 pmol each) according to manufacturer's instructions. The reaction was then
denatured for 5 minutes at 95°C and separated on a 6% 8 M urea
polyacrylamide gel (40% solution with acrylamide : bis-acrylamide = 19:1) in 1x
TBE (1.78 M Tris, 1.78 M borate, 40 mM EDTA pH 8.0) at 300 V (Sambrook et
al., 1989). Next, the RNAs were electro-blotted in a Biorad Trans blot cell onto
Hybond-N membrane (Amersham Pharmacia Biotech) and hybridised with 32P
γ-labelled oligodeoxyribonucleotide PGK3UTRrev as described in 2.4.2.
2.5 Protein methods
2.5.1 Luciferase assay and protein quantification Extracts were prepared from cells grown in YNB medium to OD600 0.8-0.9
following the protocol in Oliveira et al. (1993b). 3 ml of each yeast culture were
pelleted by centrifugation (860 x g, 5 minutes) and washed twice with ice-cold
50 mM Tris.Cl pH 7.5. Resuspended in 300 µl of this buffer, the cells were
broken in three rounds of consecutive vortexing with glass beads (30 seconds)
and cooling on ice (1 minute). The cell debris was separated from the
supernatant by centrifugation at 4°C and 16,100 x g for 5 minutes. Luciferase
activity in these lysates was measured using standard procedures (de Wet et
al., 1987; Tatsumi et al., 1988). 10 µl of the supernatant was mixed with 350 µl
reaction buffer (25 mM glycylglycine pH 7.8, 5 mM ATP pH 7.5, 15 mM MgSO4)
in a test tube. Measurement of luminoscence was for 10 seconds and initiated
by the injection of a luciferin solution (0.2 mM D-luciferin in 25 mM glycylglycine
pH 7.8) in a luminometer (Berthold Lumat LB 9507).
69
2. Materials and Methods
The total protein content per sample was estimated using a commercial kit
(Sigma) based on the biuret reaction (Smith et al., 1985). 100 µl of the sample
were mixed with 900 µl of the assay solution (50 volume parts BCA-Solution : 1
volume part 4% Copper(II)-sulphate in water) and incubated at 37°C for 30
minutes. The protein concentration was determined by comparing the OD562 of
this sample with the absorbance of standards prepared with known quantities of
BSA.
2.5.2 Western blotting For the purpose of Western blotting, 15 µg of proteins isolated by cell lysis with
glass beads (see 2.5.1) were separated by SDS polyacrylamide gel
electrophoresis (PAGE) according to Laemmli (1970). Separation was on a
12.5% polyacrylamide gel (30% solution with acrylamide : bis-acrylamide =
29:1) using the BioRad Mini Protean II system and a glycine buffer (25 mM
Tris.Cl pH 8.6, 192 mM glycine, 0.15% SDS). Prior, samples were denatured for
5 minutes at 95°C in sample buffer (final concentrations of 15.6 mM Tris.Cl pH
6.8, 5% glycerol, 0.4% SDS, 0.004% ß-mercaptoethanol, 0.001% bromophenol
blue). Runs were performed at 180 V until the bromophenol dye was 1 cm
above the bottom of the gel.
Transfer of proteins to a Hybond-P membrane (Amersham Pharmacia
Biotech) was in a Biorad semi-dry trans blot cell. Before, the membrane was
equilibrated for 10 minutes in methanol and transfer buffer (2.5 g/l glycine, 5.8
g/l Tris, 0.37 g/l SDS, 20% (v/v) methanol) each. Gel and membrane were
sandwiched between 4 sheets of Whatman paper (3M) on top and bottom; the
transfer took 30 minutes at 10 V and 400 mA.
Afterwards, the membrane was blocked by shaking in TBS (80 g/l NaCl,
2.2. g/l KCl, 18.2 g/l Tris pH 7.8) with 5% milk powder for 30 minutes. Incubation
with the rabbit anti-Flag antibody was in the cold room (4°C) over night in 2.5%
TBS. After three washes with TBS for 5 minutes at room temperature, the
second antibody (anti-rabbit conjugated with alkaline phosphatase) could bind
to the membrane in TBS for one hour at room temperature with shaking. The
membrane was washed as before and developed in the dark room with 10 ml
50 mM NaHCO3 pH 9.6, 33 µl 50 mg/ml BCIP (in 70% DMF) and 66 µl 50 mg/ml
NBT (in 100% DMF).
70
2. Materials and Methods
2.6 Polysomal gradient analysis Plasticware, glassware and solutions (were applicable) were treated with DEPC
to guarantee RNA integrity throughout the analysis. In a procedure adopted
from Sagliocco et al. (1993), cells were grown in 150 ml YNB to an OD600 of 0.5-
0.6, at which translation was stopped by addition of cycloheximide to 100 µg/ml.
The culture was divided into 50 ml Falcon tubes and cooled on ice for 5
minutes. Then, cells were collected at 1,240 x g for 5 minutes, washed once
with 10 ml breaking buffer (10 mM Tris.Cl pH 7.4, 100 mM NaCl, 30 mM MgCl2,
100 µg/ml cycloheximide) and resuspended in 600 µl breaking buffer. The cells
were broken with an equal volume of glass beads in 6 rounds of 30 seconds
vortexing and 30 seconds cooling on ice. After separation of the cell debris by 5
minutes centrifugation at 16,100 x g and 4°C, the supernatant was layered onto
a 17%-47% sucrose gradient. This gradient was prepared in a Beckman
ultracentrifugation tube (No 331374) using a standard continuous gradient mixer
and 6 ml of each DEPC-treated 17% and 47% sucrose solution (in 50 mM
Tris.Cl pH 7.4, 50 mM NH4Cl, 12 mM MgCl2, 1 mM DTT). Centrifugation was for
3.5 hours at 270,000 x g in a SW 40Ti swing-out rotor.
The gradient was then immediately fractioned in a density gradient
fractionator (ISCO model 185) with a UV flow-through absorbance detector
(ISCO UA-5) using 50% sucrose (in DEPC-treated water). The RNA of 0.6 ml
fractions was instantly extracted in two rounds of
phenol:chloroform:isoamylacohol (25:24:1, v/v) extractions and precipitated with
0.3 M sodium acetate in ethanol. For mRNA detection in the respective
fractions, the complete RNA sample was subjected to the Northern blotting
procedure described in 2.4.1.
2.7 In vitro translation in cell-free yeast extracts Template preparation by plasmid digestion or PCR amplification
For transcription, the CsCl-purified pHST7-derived templates were digested with
NsiI according to manufacturer's instruction. After control for complete
restriction on a 1% agarose gel (Sambrook et al., 1989), the ends were filled up
with the Klenow polymerase fragment at 37°C for 30 minutes following the
producer's protocol. Purification from proteins was by phenol (pH 8.0),
phenol:chloroform:isoamylacohol (P:C:I = 25:24:1, v/v) and C:I (25:24, v/v)
71
2. Materials and Methods
extraction. The DNA was precipitated with 0.3 M sodium acetate in ethanol,
salts removed by washing with 70% ethanol, and the dried DNA pellet was
resuspended to give a final concentration of approximately 1 µg/µl, which was
controlled by agarose electrophoresis and UV visualisation together with a DNA
sample of known concentration.
To obtain transcripts comprising the T7 promoter but lacking the 30
adenosine stretch of pHST7, templates were raised from plasmids pHST7-FL
and pHST7-FL-39-IRE by PCR amplification with primers pHS1-F and P3XM-R.
These products were purified from the original plasmids using the PCR
purification kit from Qiagen.
In vitro transcription
Templates were transcribed with T7 polymerase (NEBL) according to
manufacturer's specifications in the presence of cap-analogue m7GpppG for 2
hours at 37°C. After incubation with DNase and RNasin in compliance with
manufacturer's specifications, DNA degradation was controlled by agarose
electrophoresis.
The transcripts were separated from proteins and free nucleotides by
phenol/chloroform/isoamylacohol extractions and precipitation with ammonium
acetate followed by gel filtration (NAP-5 column, Pharmacia) and another round
of precipitations with ammonium acetate and sodium acetate.
Preparation of cell-free extracts
Cell-free translation extracts were prepared from diploid yeast strain W303
transformed with either the mock plasmid YCpSupex (Oliveira et al., 1993b) or
YCpSUP-IRP1. Preparation from cells grown in 2x 400 ml YNB galactose to
OD600 0.8 followed the general procedures described (Gasior et al., 1979;
Gerstel et al., 1992; Iizuka and Sarnow, 1997). The cells were cooled for 20
minutes on ice, collected at 1,240 x g for 5 minutes, and the cell pellet was
washed twice with ice-cold sterile water. In 50 ml tubes, the cells were then
washed three times in Buffer A (30 mM HEPES/KOH pH 7.4, 100 mM KAc, 2
mM MgAc) with 85 g/l mannitol by centrifugation as before. The cell pellet was
dried and resuspended in the cold room in 1.5 ml buffer A with mannitol for
each gramm cell pellet. Phenylmethyl-sulphonyl fluoride (PMSF) was added to
1 mM and glass beads (treated with HCl and extensively washed) to 3 g per g
72
2. Materials and Methods
pellet. The cells were broken manually in 5-6 cycles of 1 minute vigorous
shaking and 1 minute cooling on ice. The debris was separated by two
centrifugations at 4°C and 35,000 x g for 5 and 10 minutes, respectively. 4 ml of
the supernatant was loaded onto a GF25 column equilibrated with buffer A and
1 mM PMSF. The samples were collected in 1.5 - 2 ml fractions, assayed for
OD260/OD280, and translational activity was measured as described below. 80 µl
aliquots of active fractions were snap-frozen in liquid nitrogen and stored at -
80°C.
In vitro translation
In vitro translation was for 60 minutes at 25°C with 15 µl of cell-free extract and
the described amount of capped RNA in a final volume of 30 µl. The reaction
buffer contained final concentrations of 22 mM HEPES pH 7.4, 120 mM KOAc,
2 mM MgOAc, 0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate, 1.7 mM
DTT, 0.4 mg/ml creatine kinase, 1 U/µl Rnasin and 2 µl / 30 µl amino acid
mixture complete (Promega). The reaction was stopped by incubation on ice,
luciferase activity was measured in duplicate as described (see 2.5.1).
2.8 Reproducibility All experiments shown have been done at least three times with similar results,
and results of representative experiments are shown together with mean values
± standard deviation (STD). To correct the protein-content specific luciferase
activity (V1) for luc mRNA abundance (V2), the overall mRNA-specific translation
efficiency (V12) was calculated as V12 (± STD12) = V1 (± STD1) / V2 (± STD2) with
STD12 = ((STD1/V1)2+(STD2/V2)2))0.5.
73
3. Results
3. RESULTS
3.1 Ribosome recycling: 3'-UTR accessibility and translational efficiency
3.1.1 The potential role of 3'-UTR accessibility for ribosomal recycling on a circular mRNA
In the closed loop model of the polysome, the 5' and the 3'-ends of the mRNA
work together to enhance translational efficiency (see also Section 1.2.5). Due
to the vicinity of termination and initiation site, only interrupted by the 5'-UTR
and 3'-UTR, respectively, circularisation of the mRNA could facilitate the
intramolecular recycling of ribosomes. In one model, ribosomes are thought to
be guided back to the 5'-end via the bridging afforded by the proposed
interaction chain 3'-UTR – poly(A) tail – PABP – eIF4G. Blocking the access of
the ribosome to part or all of the 3'-UTR should interfere with posttermination
scanning and 'channelling' to the cap, and thus inhibit reinitiation within the
circular mRNP structure. We therefore devised experiments that would allow us
to modulate the behaviour of ribosomes downstream of termination.
3.1.2 Structures in the 3'-UTR can reduce translational efficiency As outlined in Section 1.2.4.4, the binding of IRP1 to IRE sequences is an ideal
and inducible system to establish a stable secondary structure on any position
within a transcript. Whereas the IRE itself only forms a very weak stem-loop and
can be resolved easily by scanning or translating ribosomes, IRP1-binding to
IRE is very efficient and forms a stable complex (Oliveira et al., 1993a).
Koloteva et al. (1997) showed this complex to impede efficiently ribosomal 40S
scanning along the mRNA when the IRE is located cap-distal. Moreover, stable
secondary structures (other than IRE-IRP1) have also been demonstrated to
block posttermination scanning 3' of an uORF (Vilela et al., 1999).
Previous studies (Koloteva et al., 1997; Oliveira et al., 1993a; Linz et al.,
1997), reported a 90% inhibition of translation upon IRP1-binding to an IRE
located 9 nt into the 5'-UTR of a luciferase reporter mRNA, most likely by
preventing the 43S preinitiation complex from binding to the cap structure. This
construct, FL-5'IRE (originally termed IRE-wt in Oliveira et al., 1993a; see also
table 2.5), was used as a control construct to ensure reproducibility of
74
3. Results
repression by IRP1 in the S. cerevisiae strain (diploid W303) and under the
growth conditions used (see Material and Methods). We employed the bipartite
system established by Oliveira et al., 1993a, whereby in a yeast double
transformant arrangement, the IRP1 ORF was placed under the control of an
galactose-inducible promoter (GAL1-PGK1 fusion promoter, GPF, Oliveira et
al., 1993b), and the IRE on a luciferase reporter mRNA that is expressed
constitutively by the TEF1 promoter (Figure 3.1A; Vega Laso et al., 1993).
To determine whether restriction of the 3'-UTR accessibility by IRE-IRP1
structures affects translational activity of a luciferase reporter mRNA in vivo,
constructs with an IRE sequence located downstream of the LUC termination
codon were assessed for their luciferase activity. In the onset of our work we
considered that inserting the IRE at several positions relative to the stop codon
might affect termination and posttermination behaviour of the ribosomes
differently (see Figure 3.1B, Table 2.5). In construct FL-15-IRE, the IRE stem-
loop is sufficiently close (15 nt) for the IRE-IRP1 complex to disturb a
terminating ribosome covering 16 nt downstream of the termination site as
shown by primer extension experiments in Wang et al. (1999). In contrast, the
mRNA of FL-24-IRE leaves enough space between termination site and IRE-
IRP1 for ribosomes to terminate properly, but migration into the 3'-UTR is
restricted. In FL-39-IRE, ribosomes could scan shortly along the 3'-UTR but
would soon be blocked 5' of the induced complex. In FL-141-IRE, the IRP1
target sequence was inserted in duplicate 141 nt downstream of the LUC stop
codon and 37 nt upstream of the poly(A) tail (Table 2.5 and Figure 3.1B).
Initially derived as a cloning by-product, the tandem IRE sequences are
expected to recruit two IRP molecules to the 3'-UTR. They are therefore
predicted to impose a very efficient blockage to potential 3'-UTR scanning when
ribosomes would almost have reached the poly(A) tail, potentially leading to
'queuing' of ribosomes (or subunits).
Taking luciferase activity of the IRE-deficient plasmid FL as a reference
value, co-expression of the human IRP1 gene led to differing degrees of
inhibition, depending on the position of the IRE. For easier comparison, the
luciferase values from cells harboured in either glucose or galactose (Figure
3.1C) are presented as GAL/GLU quotient (Figure 3.1D). Translational activity
is depicted as LUC values corrected for the protein content of the sample only
75
3. Results
Figure 3.1. Repression of translationvia IRP1-binding to the 3'-UTR. (A) TheYCpSUP-IRP1 plasmid contains thehuman IRP1 ORF under the control of thegalactose-inducible GAL-PGK fusionpromoter (PGPF). The YCp22FL1 (FL)plasmid carries the firefly luciferasereporter gene LUC downstream of theconstitutive TEF1 promoter PTEF1. A =AflII, B = BamHI, C = ClaI, E = EcoRI, H= HindIII, N = NdeI, Xb = XbaI, Xh = XhoI.(B) mRNA maps of FL-derived luciferaseconstructs containing an IRE hairpin-loopin either the 5' or 3'-UTR. Numbersindicate lengths in nucleotides, theindicated IRE stem-loop covers 29 nt. (C)Relative luciferase activities arepresented for both non-induced (glucose,GLU) and induced (galactose, GAL)conditions. The values are normalised toFL and corrected for the protein contentof the sample.
52%
69%
63%
100%
100% 10
%
46%
74%
72%
107%
100% 5%
0%
25%
50%
75%
100%
125%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
E
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
108%
112%
102%
106%
97%
100%
97%
98%
93%
110%
99%
100%
0%
25%
50%
75%
100%
125%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
E
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
co-transformed with mock plasmid Supex2
YCpSup-IRP1 CEN4 ARS1 URA3PGFP
BH C XbA H
IRP1 PGK1 terminator
Xh
YCp22FL1 CEN4 ARS1 TRP1LUCPTEF1
BC Xb/EA N H
PGK1 terminator
Xh
A
LUC An167
LUC An167
LUC37
LUC An
LUC An
LUC An167
FL-24-IRE
FL-39-IRE
FL-141-IRE
FL
FL-5’IRE
FL-15-IRE
B
E
D
F
FL FL-5
’IRE
FL-1
5-IR
E
FL-2
4-IR
E
FL-3
9-IR
E
FL-1
41-IR
E
glucoseLUC
PGK1
100±11
100±17
93±19
100±6
106±8
86±24
LUC/PGK1[%] ± STD
C
100% 87%
91%
81%
116%
92%
60%
101%
65%
114%
126% 11
%
0%
25%
50%
75%
100%
125%
150%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
ERel
ativ
e Lu
cife
rase
Act
ivity
co
rrec
ted
for p
rote
in c
onte
nt o
nly GLU
GAL
galactoseLUC
PGK1
100±8
94±20
208±23
86±16
84±21
133±12
LUC/PGK1[%] ± STD
An15
153
153
24
39
141 27
52%
69%
63%
100%
100% 10
%
46%
74%
72%
107%
100% 5%
0%
25%
50%
75%
100%
125%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
E
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
108%
112%
102%
106%
97%
100%
97%
98%
93%
110%
99%
100%
0%
25%
50%
75%
100%
125%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
E
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
108%
112%
102%
106%
97%
100%
97%
98%
93%
110%
99%
100%
0%
25%
50%
75%
100%
125%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
E
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
co-transformed with mock plasmid Supex2
YCpSup-IRP1 CEN4 ARS1 URA3PGFP
BH C XbA H
IRP1 PGK1 terminator
Xh
CEN4 ARS1 URA3PGFP
BH C XbA H
IRP1 PGK1 terminator
Xh
YCp22FL1 CEN4 ARS1 TRP1LUCPTEF1
BC Xb/EA N H
PGK1 terminator
Xh
CEN4 ARS1 TRP1LUCPTEF1
BC Xb/EA N H
PGK1 terminator
Xh
A
LUCLUC An167
LUCLUC An167
LUCLUC37
LUC AnLUCLUC An
LUC AnLUCLUC An
LUCLUC An167
FL-24-IRE
FL-39-IRE
FL-141-IRE
FL
FL-5’IRE
FL-15-IRE
B
E
D
F
FL FL-5
’IRE
FL-1
5-IR
E
FL-2
4-IR
E
FL-3
9-IR
E
FL-1
41-IR
E
glucoseLUC
PGK1
100±11
100±17
93±19
100±6
106±8
86±24
100±11
100±17
93±19
100±6
106±8
86±24
LUC/PGK1[%] ± STD
C
100% 87%
91%
81%
116%
92%
60%
101%
65%
114%
126% 11
%
0%
25%
50%
75%
100%
125%
150%
FL
FL-5'IR
E
FL-15-IR
E
FL-24-IR
E
FL-39-IR
E
FL-141-IR
ERel
ativ
e Lu
cife
rase
Act
ivity
co
rrec
ted
for p
rote
in c
onte
nt o
nly GLU
GAL
galactoseLUC
PGK1
100±8
94±20
208±23
86±16
84±21
133±12
LUC/PGK1[%] ± STD
An15
153
153
24
39
141 27
(D) Ratios of luciferase activities with and without induction, corrected for either protein content of thesample only or for both protein content and the relative LUC mRNA abundance in the sample. (E) Therelative mRNA abundance data was derived from steady-state Northern blots probed for LUC and PGK1mRNAs. LUC abundance is expressed as LUC/PGK1 ratio, the averaged values are given in percent ±standard deviation and are normalised to FL. (F) Control experiment assaying luciferase activities of cellsco-transformed with the mock plasmid Supex2 expressing no ORF upon culturing in galactose. The valuesare normalised to FL and corrected for either protein content of the sample only or for both protein contentand the relative LUC mRNA abundance in the sample (Northern blot not shown).
76
3. Results
or for both the protein content and the LUC mRNA abundance. In accordance
with the respective publications (Koloteva et al., 1997: Linz et al., 1997; Oliveira
et al., 1993b) this illustrates the effect of transcript stabilisation upon mRNA-
specific translational efficiency. The difference between these two values
therefore allows locating the reason for the change in LUC activity, e.g. whether
a reduction of the transcript abundance causes an observed decrease in gene
expression.
The control construct FL-5'IRE showed a repression of about 95% upon
IRP1 expression after correction for protein and RNA levels (Figure 3.1D). This
degree of inhibition is in agreement with previous measurements using this
construct (Oliveira et al., 1993b) and therefore confirms the effectiveness of the
experimental set-up. Where the IRE was sufficiently close to the stop codon to
allow terminating ribosomes to destabilise this element's structure (FL-15-IRE),
there was no significant effect on translation (Figure 3.1D). In earlier work,
Niepel and colleagues (1999) found that positioning a very stable (43-base pair)
stem-loop 4 nucleotides downstream of the stop codon of a LUC mRNA
resulted in the loss of half of the measurable luciferase activity. However, these
results were generated from experiments with mRNA electroporated into yeast
spheroblasts, and are therefore not directly comparable with the ones presented
here. It is also important to note that the stem-loops used by these authors were
far more stable than the IRE structure, which were shown previously to be
readily destabilised by even a scanning yeast ribosome (Oliveira et al., 1993a).
In the case of FL-15-IRE, therefore, a terminating ribosome would be expected
to be readily capable of disrupting or occluding an IRE element, thus preventing
inhibitory effects that might be associated with IRP1-binding.
After correction for protein and mRNA abundance in the sample, 28% and
26% inhibition was measured when the IRE was moved further 3' (FL-24-IRE
and FL-39-IRE, respectively). The IRP1 binding sites of both constructs allow
proper termination, and the positioning of IRE 15 nt further downstream in FL-
39-IRE seems to be without additional effect on translation. Locating two IRE
elements close to the site of polyadenylation (FL-141-IRE) led to approximately
50% inhibition of translation upon IRP1-binding (Figure 3.1D). The latter
construct recruits two IRP proteins to the 3'-UTR and potentially establishes a
more stable structural barrier. However, the observed enhanced repression of
77
3. Results
translation is more likely to be position-dependent since a poly(G) tract at this
very position resulted in a comparable decrease in LUC activity (see below).
Growth in carbon sources different from glucose is known to change the
translational pattern in S. cerevisiae (Kuhn et al., 2001); moreover, IRP1-
binding to IRE could disturb protein interactions with the 3'-UTR. The first issue
was addressed by co-transforming cells either with a 'mock' plasmid (Supex2)
containing no inducible ORF. By comparing LUC expression after growth in
glucose and galactose, all constructs were found to be expressed to the same
extent and without change in mRNA stability (Figure 3.1F). In a different control
experiment, the IRE-IRP1 system and thus the necessity of harbouring the cells
in galactose was circumvented. For this, constructs with an intrinsic secondary
structure consisting of a 18 guanosine stretch close to the stop codon (FL-6-pG)
or further downstream (FL-131-pG) were created (Figure 3.2A). Muhlrad et al.
(1995) showed that this poly(G) tract inserted into the 5'-UTR can inhibit
translation and stabilise PGK1 mRNA. Indeed, a position-dependent inhibitory
effect similar to the one with IRE-bearing constructs was observed with these 3'-
UTR located poly(G) tracts, although repression was not as effective as with
binding of IRP1 to the mRNA (Figure 3.2B).
The luciferase assay results indicate that the insertion of a stable
obstruction in the 3'-UTR imposes a position-dependent effect on translation.
Whereas the terminating ribosome can obviously destabilise the IRE-IRP1
interaction, placing the complex further downstream reduces the translation
activity of the mRNA although the effect is not as pronounced as with a 5'-UTR
located structure. The effect is more evident when two IRP1 molecules bind to a
site close to the poly(A) tail. With reference to the lower extent of translational
repression, it can be concluded that 3'-UTR scanning is not an absolute
requirement for the posttermination ribosome to be able to recycle and
contribute to the overall translational efficiency of a specific mRNA. mRNA
abundance was calculated from Northern blots showing the steady-state levels
of the LUC transcript and the reference mRNA PGK1 (Figure 3.1E, Figure
3.2.C). Two-fold stabilisation of FL-5'IRE upon IRP1-binding was observed
previously by Koloteva et al. (1997) and Linz et al., (1997) and was interpreted
to be an effect of translational inhibition of the luciferase transcripts. The same
result has been seen with intrinsic structural elements e.g. stem-loops of the
78
3. Results
mRNA (Oliveira et al., 1993b). In agreement with this finding is the observation
that translational repression of the 3'-UTR modified constructs FL-131-pG and
FL-141-IRE was coupled with mRNA stabilisation. The smaller increase in
transcript stability – when compared to FL-5'IRE – also correlates to the lesser
extent with translation repression of the 3'-UTR modified constructs.
3.1.3 Recruitment of IRP1 to the 3'-UTR modulates translation and not polyadenylation
3.1.3.1 poly(A)-length and -site verification The insertion of the IRE sequences and poly(G) tracts was supplementary to
the 3'-UTR sequence, i.e. they did not replace any parts of the trailer that might
function as binding sites for 3'-UTR specific factors. Moreover, the insertion
sites were carefully planned to avoid disturbance of the poly(A) signals
(compare sequences in Table 2.5). However, changes in the structure of the 3'-
UTR such as by IRP1-binding could theoretically influence the state of
adenylation of the mRNA, which in turn could modulate translation initiation and
mRNA stability. Therefore, I examined the average length of the poly(A) tails on
the IRE-bearing mRNAs from cell extracts with IRP1 being co-expressed.
LUC An
LUC An131 ntG18
LUC An6 ntG18
FL
FL-6-pG
FL-131-pG
C
LUCPGK1
FL FL-6
-pG
FL-1
31-p
G
92±8
100±7
LUC/PGK1[%] ± STD
117±9
A72
%
98%
100%
62%
107%
100%
0%
25%
50%
75%
100%
125%
FL
FL-6-pG
FL-131-pG
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected forBLUC An
LUC An131 ntG18
LUC An6 ntG18
FL
FL-6-pG
FL-131-pG
LUC An
LUC An131 ntG18
LUC An6 ntG18
LUC AnLUC An
LUC An131 ntG18LUC An131 ntG18G18
LUC An6 ntG18LUC An6 ntG18G18
FL
FL-6-pG
FL-131-pG
C
LUCPGK1
FL FL-6
-pG
FL-1
31-p
G
92±8
100±7
LUC/PGK1[%] ± STD
117±9
LUCPGK1
FL FL-6
-pG
FL-1
31-p
G
92±8
100±7
LUC/PGK1[%] ± STD
117±9
A72
%
98%
100%
62%
107%
100%
0%
25%
50%
75%
100%
125%
FL
FL-6-pG
FL-131-pG
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected for
72%
98%
100%
62%
107%
100%
0%
25%
50%
75%
100%
125%
FL
FL-6-pG
FL-131-pG
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected forB
Figure 3.2. Position-dependent decrease of translational efficiency by a 3'-UTR-locatedpoly(G) tract. (A) mRNA maps of FL and derived constructs carrying a poly(G) stretch in the 3'-UTR. (B) Relative luciferase activities of poly(G)-bearing constructs, normalised to FL andcorrected for either protein content of the sample only, or for both protein content and the relativeLUC mRNA abundance in the sample. (C) Typical steady-state Northern blot of extracts fromstrains carrying the respective constructs, probed for LUC and PGK1 mRNAs. The averagedvalues for relative LUC mRNA abundance are given under the respective lanes of the Northernblot.
79
3. Results
RNase H treatment of mRNAs extracted from cells expressing IRP1 was used
to cause cleavage either at the end of the LUC ORF alone, or at the end of the
LUC ORF and within the poly(A) tail simultaneously. The size of the two 3'
fragments was discernible after separation on a 6% polyacrylamide gel and
corresponds to the average in vivo poly(A) length of each mRNA. In a Northern
blot hybridised with a 3'-UTR targeted 32P-labelled oligodeoxyribonucleotide
A
LUC AAAAAn
poly(T)
LucRev
200
500
400
300
600
+ +
FL+
+ +
FL-1
5-IR
E+
+ +
FL-2
4-IR
E+
+ +
FL-3
9-IR
E+
+ +
FL-1
41-IR
E+LucRev
poly(T)M
in nt
poly(A) tailpoly(A) site3'UTRB
AAAAAn
3'UTR only
3'UTR + poly(A)
A
LUC AAAAAnLUC AAAAAn
poly(T)
LucRev
200
500
400
300
600
+ +
FL+
+ +
FL-1
5-IR
E+
+ +
FL-2
4-IR
E+
+ +
FL-3
9-IR
E+
+ +
FL-1
41-IR
E+LucRev
poly(T)M
in nt
200
500
400
300
600
200
500
400
300
600
+ +
FL+
+ +
FL-1
5-IR
E+
+ +
FL-2
4-IR
E+
+ +
FL-3
9-IR
E+
+ +
FL-1
41-IR
E+LucRev
poly(T)M
in nt
poly(A) tailpoly(A) site3'UTRB
AAAAAn
3'UTR only
3'UTR + poly(A)
Figure 3.3. Binding of IRP1 to an IRE located in the 3'-UTR does not affectpolyadenylation. (A) Total RNA extracted from each yeast strain co-expressing the IRP1 geneand one of the LUC constructs was subjected to RNase H treatment in the presence ofspecifically targeted oligodeoxyribonucleotides. Incubation with the oligodeoxyribonucleotideLucRev led to digestion upstream of the LUC stop-codon, while the further addition of theoligodeoxyribonucleotide poly(T) caused degradation of the poly(A) tail as well. The lengthdifference of the single (white arrows) and double digestion products (black arrows) detected inNorthern blots by probing for a 3'-UTR sequence corresponds to the poly(A) tail length. Thelengths (in nucleotides) of RNA markers are indicated on the right hand side. (B) Dideoxyfluorescent derivative sequencing of RT-PCR products confirms that the wild typepolyadenylation site is utilised in all of the IRE-bearing constructs. The representative sequenceshown corresponds to the polyadenylated 3'-UTR of construct FL-39-IRE, co-expressed in thecell with IRP1.
80
3. Results
comparison with IRE-free construct FL revealed that neither IRE-insertion nor
IRP1-binding to its target sequence affected the distribution of poly(A) tail
lengths at any of the positions examined (Figure 3.3A). The average poly(A)
length of about 70 nt observed correlates to those published for yeast mRNAs
(Brown & Sachs, 1998).
Modifications of the 3'-UTR sequence could not only affect poly(A) tail
length but also change the choice of the poly(A) site (Düvel et al., 1999). Thus,
LUC mRNAs extracted from cells expressing IRP1 were subjected to first strand
synthesis and subsequent PCR amplification of their 3'-UTR. Evidence was
then obtained via sequencing of the RT-PCR products that utilisation of the wild
type polyadenylation site was unaffected (Figure 3.3B).
3.1.3.2 Polysomal analysis Efficiently translated mRNAs are bound to more than one ribosome in a
structure called the polysome. For further investigation of the change in the
overall translational state conferred by IRP1-binding to the 3'-UTR, reference
construct FL, control construct FL-5'IRE and two mRNAs with the IRE in the 3'-
UTR – FL-39-IRE and FL-141-IRE – were subjected to sucrose gradient
analysis. As described in Material and Methods, extracts of cells expressing the
respective construct in the presence of IRP1 were separated by centrifugation
in a sucrose gradient, which was analysed thereafter for absorbance at 260 nm.
From the resulting graph (Figure 3.4A), the peaks corresponding to 40S (43S),
80S, di-, tri- and further polysomes can be derived.
As indicated by the representative polysomal profile of construct FL-141-
IRE, overall translation in strain W303 is not impaired by the growth in
galactose. Most ribosomes are not dissociated in 40S and 60S subunits but
present in one or more copies per mRNA. The respective sucrose fractions
were then assessed in Northern blots for their LUC and PGK1 mRNA
abundance (Figure 3.4B). For better visualisation, the LUC/PGK1 mRNA
abundance ratios of these blots have been normalised to the values of the
respective 80S peak and are depicted as bars in Figure 3.4C. In the Northern
blot of construct FL, most of the LUC mRNA is detectable in the polysomal
fractions, although PGK1 is translated even more efficiently (Figures 3.4B,C).
The substantial shift of FL-5'IRE mRNA towards the 40S / 43S and monosomal
fractions can be interpreted as a result of IRP1-binding to an IRE close to the
81
3. Results
cap. The strong inhibition of 80S formation and therefore, translation initiation is
in agreement with observations by Koloteva et al. (1997) and Linz et al. (1997).
With IRP1 being co-expressed, the major part of both, FL-39-IRE and FL-
141-IRE mRNAs, was localised in the lower polysomal or 80S fraction (Figure
3.4B,C). Moreover, the abundance of transcripts in the polysomal fractions
matches the degree of IRP-conferred repression of luciferase activity observed
for the individual constructs (FL-39-IRE: 74%, FL-141-IRE: 47% as in Figure
fraction
5‘IRE
FL-141-IRE
FL-39-IRE
FL
B101 2 3 4 5 6 7 8 9
PGK1
LUC
PGK1
LUC
PGK1
LUC
PGK1
LUC
A
–40
S
–60
S–
80S
OD
260
––––
–43
S
polysomes
1 2 3 4 5 6 7 8 9 10
FL-141-IRE
FL-39-IRE
FL-5'IREFL
0%
100%
200%
300%
400%
Rel
ativ
e LU
C/P
GK
1 m
RN
AA
bund
ance
polysomal fraction
C
fraction
5‘IRE
FL-141-IRE
FL-39-IRE
FL
B101 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9
PGK1
LUC
PGK1
LUC
PGK1
LUC
PGK1
LUC
A
–40
S
–60
S–
80S
OD
260
––––
–43
S
polysomes
–40
S
–60
S–
80S
OD
260
––––
–43
S
polysomes
1 2 3 4 5 6 7 8 9 10
FL-141-IRE
FL-39-IRE
FL-5'IREFL
0%
100%
200%
300%
400%
Rel
ativ
e LU
C/P
GK
1 m
RN
AA
bund
ance
polysomal fraction
C
Figure 3.4. IRP1-binding toIREs in the 5' or 3'-UTR affectsthe polysomal distribution ofLUC mRNA. Sucrose gradientanalysis was performed onextracts from cells containing aLUC reporter construct afterinduction of IRP1 synthesis for12 hours in galactose medium.(A) The diagram shows thefractional distribution of opticaldensity at 260 nm in a sucrosegradient. The representativeprofile was derived from cell-extracts containing FL-141-IRE.Fractions corresponding tomRNA bound to 40S/43S, 60S or80S ribosomes are indicated. (B)Northern blots of the numberedfractions as indicated, theluciferase construct probed for isspecified alongside each panel.Blots were co-hybridised with aPGK1-specific probe to revealthe polysomal distribution of achromosomally encoded controlmRNA. (C) Diagram illustratingthe relative abundance of LUC toPGK1 mRNA among thepolysomal fractions. For bettercomparison, the values arenormalised to the magnitude ofthe respective 80S peak (fraction/ lane 4).
82
3. Results
3.1D). The reference mRNA PGK1 is present in the high-molecular fractions for
all the luciferase constructs tested (Figure 3.4B).
These alterations of the polysomal profile can be taken as additional
evidence that IRP1 recruitment downstream of the stop codon changes the
function of the mRNA's translational status in a position-dependent manner.
3.1.4 Translation of a full-length ORF is insensitive to changes of the codon context
As discussed in Section 1.4, translation termination at a short uORF is sensitive
to the stop codon context (Grant & Hinnebusch, 1994a). By modulation of
ribosome release from the termination site, the necessity of downstream
scanning for efficient LUC translation can be investigated. Accordingly, the
FL
XhoI -2 -1 stop XbaIC TCG AGC AAA TTG TAA TCTAGAC TCG AGC AAA CCG TAA TCTAGAC TCG AGC CCG TTG TAA TCTAGAC TCG AGC CCG CCG TAA GCG GTT ACC TCTAGAC TCG AGC AAA TTG TAA GCG GTT ACC TCTAGAC TCG AGC AAA TTG TAA TAA TCTAGA
LUC An
C
A
FL*
-1Pr
o
-2Pr
o
-1,2
Pro1
0ds
10ds
stop
stop
LUC
PGK1
112±9
100±5
LUC/PGK1[%] ± STD
86±12
98±10
89±9
96±6
FL*-1Pro-2Pro
-1,2Pro10ds10ds
stopstop
101%
95%
103%
94%
100%
98%
100%
109%
88%
105%
105%
106%
0%
25%
50%
75%
100%
125%
FL*
-1Pro
-2Pro
-1,2P
ro10
ds10
ds
stops
top
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content only protein and RNA contentcorrected forB
FL
XhoI -2 -1 stop XbaIC TCG AGC AAA TTG TAA TCTAGAC TCG AGC AAA CCG TAA TCTAGAC TCG AGC CCG TTG TAA TCTAGAC TCG AGC CCG CCG TAA GCG GTT ACC TCTAGAC TCG AGC AAA TTG TAA GCG GTT ACC TCTAGAC TCG AGC AAA TTG TAA TAA TCTAGA
LUC AnLUC An
C
A
FL*
-1Pr
o
-2Pr
o
-1,2
Pro1
0ds
10ds
stop
stop
LUC
PGK1
112±9
100±5
LUC/PGK1[%] ± STD
86±12
98±10
89±9
96±6
FL*
-1Pr
o
-2Pr
o
-1,2
Pro1
0ds
10ds
stop
stop
LUC
PGK1
FL*
-1Pr
o
-2Pr
o
-1,2
Pro1
0ds
10ds
stop
stop
LUC
PGK1
112±9
100±5
LUC/PGK1[%] ± STD
86±12
98±10
89±9
96±6
FL*-1Pro-2Pro
-1,2Pro10ds10ds
stopstop
101%
95%
103%
94%
100%
98%
100%
109%
88%
105%
105%
106%
0%
25%
50%
75%
100%
125%
FL*
-1Pro
-2Pro
-1,2P
ro10
ds10
ds
stops
top
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content only protein and RNA contentcorrected for
101%
95%
103%
94%
100%
98%
100%
109%
88%
105%
105%
106%
0%
25%
50%
75%
100%
125%
FL*
-1Pro
-2Pro
-1,2P
ro10
ds10
ds
stops
top
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content only protein and RNA contentcorrected forB
Figure 3.5. The stop codon context of the full-length luciferase ORF does not affecttranslational efficiency or mRNA stability. (A) mRNA map of FL-derived construct FL*containing an XhoI restriction site upstream of the stop codon facilitating the change of the stopcodon context. Modifications took the form of mutations of codons at positions -1 and -2upstream of the LUC stop codon to CCG (proline), the introduction of the 10 nucleotidedownstream sequence from GCN4 uORF4, or the introduction of an additional stop codon. (B)Relative luciferase activities of stop codon context mutants, normalised to FL* and corrected foreither protein content of the sample only or for both protein content and the relative LUC mRNAabundance in the sample. (C) Typical steady-state Northern blot of FL* and derivatives, probedfor LUC and PGK1 mRNAs. The averaged values for relative LUC mRNA abundance are givenunder the respective lanes of the Northern blot.
83
3. Results
effects of changing the stop codon context so as to maximise termination
efficiency were explored.
One approach was to introduce CCG codons encoding proline into the last
two positions of the coding sequence, since these have been shown to promote
efficient translation termination (Figure 3.5A). Introduction of these codons
suppresses read-through (Mottagui-Tabar et al., 1998), most likely by affecting
peptidyl-tRNA hydrolysis rates. In a further approach, an additional stop codon
immediately after the normal luciferase gene stop codon provided the ribosome
with a second opportunity to recognise a site of peptide chain termination. The
combination of two stop codons can efficiently suppress read-through in E. coli
(Major et al., 2002). Finally, in a further construct the ten nucleotides
downstream of the stop codon were changed to those of GCN4 uORF4. The
latter downstream context is known to suppress the reinitiation competence of
terminating ribosomes (Grant and Hinnebusch, 1994a), even in the context of a
different mRNA (Vilela et al., 1999).
LUC measurements and Northern blots of mRNA steady-state levels
revealed that none of these approaches affected the specific translation rates or
the stabilities of the LUC mRNA significantly (Figures 3.5B,C). This is in
contrast to termination at a short uORF such as the ones in the GCN4 leader.
Termination there is sensitive to the stop codon context, and the G/C ratio has
been shown to modulate the balance of reinitiation and ribosomal release
(Grant and Hinnebusch, 1994a). One way to explain the observed results is to
assume that the ribosome terminating after translation of a long ORF differs
from those at the stop codon of a short uORF in its sensitivity towards the stop
codon context. Although e.g. the ribosomal reinitiation capability depends on the
ORF length (see below), there is no direct evidence supporting this assumption.
Notably, as described above, also recruitment of IRP1 to an IRE
immediately 3' of the termination site did not affect translational efficiency or
mRNA stability (see Figure 3.1D, construct FL-15-IRE). If, as it seems likely,
this and the above measures both promote release of ribosomal subunits from
the LUC mRNA, the results suggest that progression of ribosomes beyond the
stop codon into the 3'-UTR is not required for normal recycling to occur.
Alternatively, enhanced recruitment of ribosomal subunits from the free cellular
pool can compensate a (moderate) loss of intramolecular recycled ribosomes.
84
3. Results
3.1.5 The eIF4G – Pab1p interaction is not required for 3'-UTR-confered translation repression in vivo
3.1.5.1 The absence of the cap – poly(A) interaction in vivo does not abolish the translational inhibition by a 3'-UTR structure
Circularisation of mRNA by the eIF4G – Pab1p interaction requires the
presence of a poly(A) tail (Tarun and Sachs, 1996). If translational efficiency is
controlled by ribosomal 'channelling' via 3'-UTR scanning and subsequent
transfer via mentioned 5' – 3' end interaction, the disruption of the circularisation
would be expected to abolish the negative effect of 3'-UTR inaccessibility. I
therefore constructed strains, in which the impact of blocking the 3'-UTR could
be assessed against a cellular background that is defective in the eIF4G –
Pab1p interaction. For this, a mutated version of eIF4G1 (tif4631-213) in which
the binding site for Pab1p is no longer functional was obtained (Tarun et al.,
1997). This mutant was expressed from a plasmid that had been introduced into
a strain (YAS1947) in which the genes encoding eIF4G1 and eIF4G2 have both
been disrupted. Luciferase measurements revealed that despite the absence of
the eIF4G – Pab1p interaction, IRP1-binding to an IRE in the 3'-UTR still
partially repressed translation (BEFL-39-IRE; Figure 3.6A,B). The degree of
inhibition (18%) was somewhat less than that observed in the wild type
background (26%, Figure 3.1D). However, it was also observed that the degree
of inhibition caused by IRP1-binding to an IRE in the 5'-UTR was reduced in the
YAS1947 background (BEFL-5'IRE; 73% versus 95% for FL-5'IRE), which
means that the change in inhibitory potential of the IRE/IRP1 complex was not
specific to the 3'-UTR. This demonstrates that the eIF4G – Pab1p interaction is
not required for IRE-mediated modulation of translation via the 3'-UTR in vivo.
3.1.5.2 In vitro translation in cell-free extracts is not affected by IRP1-binding to the 3'-UTR
In an alternative approach to the above in vivo system, a cell-free translation
assay offered the possibility to determine whether the influence of IRE-mediated
blocking of the 3'-UTR was reproducible in vitro and could be changed by the
absence of the poly(A) tail. For this, translation competent cell extracts were
prepared from yeast cells either expressing the mock plasmid Supex2 or
YCpSup-IRP1. Aliquots were then programmed with 0-1 µg mRNA
corresponding to constructs FL, FL-5'IRE and FL-39-IRE.
85
3. Results
The analysis revealed, as reported previously (Gerstel et al., 1992; Niepel
et al., 1999; Iizuka et al., 1994), both capping (data not shown) and
polyadenylation (Figure 3.6C,D) enhanced translation of the reporter mRNA in
the cell-free system. Moreover, there was synergy between the effects of these
two modifications, so that the combined effects resulted in a sum effect that was
greater than the arithmetic sum of the individual contributions (compare Sachs,
2000). The enhanced translational activity of polyadenylated 5'-IRE mRNA in
cell extracts without IRP1 presence can be explained by the elongation of the
leader due to IRE insertion (Figure 3.6C).
97%
64%
100%
82%
27%
100%
0%
25%
50%
75%
100%
125%
BEFL
BEFL-5'IRE
BEFL-39-IRE
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for C
B
LUC
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
PGK1
galactoseglucose
110±10
100±12
LUC/PGK1[%] ± STD
123±17
133±12
91±8
315±27
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
FL FLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
104 R
LU u
nits
6
5
4
3
2
1
0
D
FLFLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
106 R
LU u
nits
5
4
3
2
1
0
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
D
A
97%
64%
100%
82%
27%
100%
0%
25%
50%
75%
100%
125%
BEFL
BEFL-5'IRE
BEFL-39-IRE
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for
97%
64%
100%
82%
27%
100%
0%
25%
50%
75%
100%
125%
BEFL
BEFL-5'IRE
BEFL-39-IRE
Rel
ativ
e Lu
cife
rase
Act
ivity
GAL
/GLU
protein content onlyprotein and RNA content
corrected for C
B
LUC
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
PGK1
galactoseglucose
110±10
100±12
LUC/PGK1[%] ± STD
123±17
133±12
91±8
315±27
LUC
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
BEF
L
BEF
L-5’
IRE
BEF
L-39
IRE
PGK1
galactoseglucose
110±10
100±12
LUC/PGK1[%] ± STD
123±17
133±12
91±8
315±27
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
FL FLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
104 R
LU u
nits
6
5
4
3
2
1
0
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.000.00 0.25 0.50 0.75 1.00
FL FLpA 39IRE 39IREpA 5'IREpA
FL FLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
104 R
LU u
nits
6
5
4
3
2
1
0
Rel
ativ
e Lu
cife
rase
Act
ivity
104 R
LU u
nits
6
5
4
3
2
1
0
D
FLFLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
106 R
LU u
nits
5
4
3
2
1
0
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
D
FLFLpA 39IRE 39IREpA 5'IREpA
D
FLFLpA 39IRE 39IREpA 5'IREpA
Rel
ativ
e Lu
cife
rase
Act
ivity
106 R
LU u
nits
5
4
3
2
1
0
Rel
ativ
e Lu
cife
rase
Act
ivity
106 R
LU u
nits
5
4
3
2
1
0
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.00
capped luciferase RNA (µg)0.00 0.25 0.50 0.75 1.000.00 0.25 0.50 0.75 1.00
D
A
Figure 3.6. Significance of 5' – 3' interactions to recycling. (A) Ratios of luciferase activitieswith and without induction, corrected for either protein content of the sample only or for bothprotein content and the relative LUC mRNA abundance in the sample. The samples wereextracted from YAS1947 cells (expressing eIF4GI-213 mutated in its Pab1p-binding) and co-transformed with YCpSUP-IRP1 and the target IRE constructs. Growth was under either non-induced (glucose, GLU) or induced (galactose, GAL) conditions. The BEFL plasmids, whichcarry an ADE2 marker, correspond to their counterparts in the FL series that have a TRP1marker. (B) Northern blots probed for LUC and PGK1 mRNAs. (C,D) In vitro transcribed andcapped mRNAs were translated in extracts derived from cells containing either (C) the controlplasmid YCpSupex without an inducible ORF or (D) YCpSUP-IRP1. The luciferase activities ofthe in vitro translation reactions are presented as titrations of added mRNA over the range 0-1µg. Open symbols represent translation data from non-polyadenylated mRNAs, while the fullsymbols correspond to mRNAs with a 30 nucleotide poly(A) tail. Bars indicate standarddeviations.
86
3. Results
However, one particular observation strongly influenced the interpretation
of the effects of downstream elements on translation in vivo. The IRP1/IRE
interaction in the 5'-UTR clearly inhibits translation as effectively as in vivo, yet
has no effect on in vitro translation in the 3'-UTR position, irrespective of the
presence or absence of a poly(A) tail (compare Figures 3.6C,D). It was also
observed that primarily 80S monosomes were formed on the reporter mRNA in
the cell-free system (private correspondence, Karine Berthelot). It follows that
the inhibitory effect of the IRP1/IRE interaction occurring on the 3'-UTR is
therefore restricted to polysomal structures in vivo.
3.2 Correlation of ORF length and ribosome reinitiation potential
3.2.1 Reinitiation capability increases as a wild type ORF is progressively truncated
Tightly connected with our initial question – whether ribosomes entering the 3'-
UTR contribute to the overall translational mRNA efficiency – is the definition of
the circumstances affecting posttermination reinitiation competence. Initially,
analysis of translational control on the GCN4 mRNA of S. cerevisiae
demonstrated that termination on a short ORF can allow ribosomes to retain
their ability to initiate translation downstream of the stop codon was (reviewed in
Hinnebusch, 1997), and this holds true for other uORFs (upstream ORFs;
McCarthy, 1998; Geballe & Sachs, 2000). It therefore can be derived that
termination on a short ORF leaves at least the 40S ribosomal subunit in a state
that must be distinct from the state that normally results from termination on the
main ORFs of genes. Since uORFs and wild type ORFs differ primarily in their
length, we needed to consider the effects of this attribute on the ribosomal
reinitiation capability.
However, only few studies have explored the effect of varying the uORF
length (Luukkonen et al., 1995; Hwang et al., 1998; Kozak, 2001), and this
research was done in mammalian and virus systems. A systematic investigation
into the relationship of reinitiation capacity and length of the upstream ORF in
yeast could not be found in the published literature. In a first step towards filling
this gap and thereby elucidating the basis of the transition from (re)initiation-
competent to (re)initiation-incompetent ribosomes that seems to occur during
87
3. Results
elongation, we studied the dependence of reinitiation capacity on the length of
the upstream ORF in a bicistronic mRNA.
For this, a FLAG-tagged, full-length actin ORF was inserted out-of-frame
upstream of luciferase in plasmid FL'. The 59 nucleotide intercistronic region
comprises the A/T-rich sequence 5'-TGTAGAATTCTAGA-3' followed by the
GCN4 uORF1 downstream sequence (see Table 2.5). The latter has been
shown to promote reinitiation on the GCN4 mRNA, probably due to its A/T
content (Grant & Hinnebusch, 1994b). Bicistronic ACT-LUC mRNAs with 3'-
truncated versions of the actin ORF were created in order to shorten the ORF to
lengths of 963 to 15 nucleotides (giving the ACTd-LUC constructs, Figure 3.7A).
The truncation procedure (see Materials and Methods section) left the
sequence context at the start codon identical for all constructs as well as the
sequence 3' of the ACT stop codon (compare Table 2.5).
As expected, downstream of a wild type full-length ORF, posttermination
initiation at the LUC ORF, and therefore LUC activity, was at a minimal level
(Figure 3.7B). Reinitiation only became readily detectably with a truncated actin
ORF of 105 nucleotides or less, whereby efficiency increased markedly with
first-ORF lengths of 63 nucleotides and shorter (Figure 3.7B). Luciferase
activities were corrected for mRNA abundance and protein, revealing a
maximum reinitiation-dependent level of luciferase (encoded by ACTd15-LUC)
equivalent to 5% of the monocistronic luciferase construct FL' (Figure 3.7B;
Table 2.5). This means that even the shortest ACT-derived ORF encoding only
four amino acids is relatively poor at promoting reinitiation on the LUC ORF.
Translation of the first ORF was confirmed by detection of the full-length FLAG-
tagged actin protein in a Western blot (Figure 3.7D). Truncated FLAG-actin
products were not readily quantifiable because of partial degradation.Constructs
that showed luciferase activity generated by reinitiation were also destabilised;
the mRNA abundance of constructs with an actin ORF of 105 nt or less was
reduced by up to 73% relative to the full-length control (Figure 3.7C). Thus,
translation termination at the ends of these shortened first ORFs partially
destabilises the mRNA, although the degree of destabilisation is smaller than
that normally associated with the nonsense-mediated decay pathway (Zhang et
al., 1995). Similar effects of uORFs on the stability of mRNAs have been
reported for other uORF-containing 5'-UTRs (Ruiz- Echevarria & Peltz, 2000;
88
3. Results
89
0.00
1%
0.02
2%
0.02
6%
0.02
0%
0.10
7%
0.00
1%
0.02
4%
0.02
6%
0.02
2%
0.28
4% 0.75
1%
0.60
4% 1.34
7%5.
006%
1.35
7% 2.00
9%
0%
1%
2%
3%
4%
5%
6%
7%
ACT-LUC
ACTd963
-LUC
ACTd672
-LUC
ACTd327
-LUC
ACTd105
-LUC
ACTd63-L
UC
ACTd33-L
UC
ACTd15-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected forB
A
C D
FL'
AC
T-LU
C
AC
Td96
3-LU
C
AC
Td67
2-LU
C
AC
Td32
7-LU
C
AC
Td10
5-LU
C
AC
Td63
-LU
C
AC
Td33
-LU
C
AC
Td15
-LU
C
ACT-LUC
PGK1 LUC
84±10
100±5
LUC/PGK1[%] ± STD
103±5
38±3
95±5
88±7
45±5
37±6
27±4
ACT-LUC
ACTd963-LUC
ACTd672-LUC
ACTd327-LUC
ACTd105-LUC
ACTd63-LUC
ACTd33-LUC
ACTd15-LUC
FL'
An
63
963
An
AnFLAG-actin luciferase170 1157 165359 153
672An
An
105An
33An
327An
15An
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
CA
CTd
963-
LUC
AC
Td67
2-LU
CA
CTd
327-
LUC
AC
Td10
5-LU
CA
CTd
63-L
UC
AC
Td33
-LU
CA
CTd
15-L
UC
0.00
1%
0.02
2%
0.02
6%
0.02
0%
0.10
7%
0.00
1%
0.02
4%
0.02
6%
0.02
2%
0.28
4% 0.75
1%
0.60
4% 1.34
7%5.
006%
1.35
7% 2.00
9%
0%
1%
2%
3%
4%
5%
6%
7%
ACT-LUC
ACTd963
-LUC
ACTd672
-LUC
ACTd327
-LUC
ACTd105
-LUC
ACTd63-L
UC
ACTd33-L
UC
ACTd15-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected for
0.00
1%
0.02
2%
0.02
6%
0.02
0%
0.10
7%
0.00
1%
0.02
4%
0.02
6%
0.02
2%
0.28
4% 0.75
1%
0.60
4% 1.34
7%5.
006%
1.35
7% 2.00
9%
0%
1%
2%
3%
4%
5%
6%
7%
ACT-LUC
ACTd963
-LUC
ACTd672
-LUC
ACTd327
-LUC
ACTd105
-LUC
ACTd63-L
UC
ACTd33-L
UC
ACTd15-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
protein content onlyprotein and RNA content
corrected forB
A
C D
FL'
AC
T-LU
C
AC
Td96
3-LU
C
AC
Td67
2-LU
C
AC
Td32
7-LU
C
AC
Td10
5-LU
C
AC
Td63
-LU
C
AC
Td33
-LU
C
AC
Td15
-LU
C
ACT-LUC
PGK1 LUC
84±10
100±5
LUC/PGK1[%] ± STD
103±5
38±3
95±5
88±7
45±5
37±6
27±4
FL'
AC
T-LU
C
AC
Td96
3-LU
C
AC
Td67
2-LU
C
AC
Td32
7-LU
C
AC
Td10
5-LU
C
AC
Td63
-LU
C
AC
Td33
-LU
C
AC
Td15
-LU
C
ACT-LUC
PGK1 LUC
84±10
100±5
LUC/PGK1[%] ± STD
103±5
38±3
95±5
88±7
45±5
37±6
27±4
ACT-LUC
ACTd963-LUC
ACTd672-LUC
ACTd327-LUC
ACTd105-LUC
ACTd63-LUC
ACTd33-LUC
ACTd15-LUC
FL'
An
63
963
An
AnFLAG-actin luciferase170 1157 165359 153
672An
An
105An
33An
327An
15An
ACT-LUC
ACTd963-LUC
ACTd672-LUC
ACTd327-LUC
ACTd105-LUC
ACTd63-LUC
ACTd33-LUC
ACTd15-LUC
FL'
An
63
963
An
AnFLAG-actin luciferase170 1157 165359 153
672An
An
105An
33An
327An
15An
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
CA
CTd
963-
LUC
AC
Td67
2-LU
CA
CTd
327-
LUC
AC
Td10
5-LU
CA
CTd
63-L
UC
AC
Td33
-LU
CA
CTd
15-L
UC
32.5
16.5
47.5
25.0
Act1p
kDa
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
CA
CTd
963-
LUC
AC
Td67
2-LU
CA
CTd
327-
LUC
AC
Td10
5-LU
CA
CTd
63-L
UC
AC
Td33
-LU
CA
CTd
15-L
UC
Figure 3.7. Truncation of the first ORF in a bicistronic ACT-LUC construct leads toincreased expression of the downstream ORF. (A) Physical maps of FLAG-tagged actin –luciferase (ACT-LUC) mRNAs. The actin ORF was progressively truncated at its 3'-end, whilethe length and sequence of the intercistronic spacer was maintained. Numbers indicate lengthsin nucleotides. (B) Relative luciferase activities of constructs, normalised to FL' (not shown) andcorrected for either protein content of the sample only or for both protein content and the relativeLUC mRNA abundance in the sample. (C) Typical steady-state Northern blot of ACT-LUC andtruncated versions, probed for LUC and PGK1 mRNAs. The averaged values for relative LUCmRNA abundance are given under the respective lanes of the Northern blot. (D) Western blotshowing expression of the actin ORF using a FLAG-antibody. The full-length FLAG-tagged actinprotein is indicated.
3. Results
Vilela et al., 1998 and 1999; McCarthy, 1998; Oliveira & McCarthy, 1995; see
Section 1.4.6 for details). A set of monocistronic control plasmids bearing the
same series of actin truncations but lacking the LUC gene was also
constructed. In the resulting mRNAs, the termination codon in each of the actin
truncation constructs was always 153 nucleotides away from the poly(A) tail
(Figure 3.8A). The abundance of these mRNAs was reduced by more than 50%
when the uORF was truncated to 63 nt or shorter (Figure 3.8B). The degrees of
destabilisation and the correlations to actin ORF length are generally similar to
the ones observed with the bicistronic constructs, although some stabilisation in
the presence of the LUC ORF is evident.
While displaying only 0.01% of the luciferase activity of the monocistronic
reference construct FL', the full-length bicistronic ACT-LUC mRNA clearly and
reproducible showed expression of the downstream ORF. Since the ORFs of all
bicistronic constructs are out of frame (see also Table 2.5), read-through of the
stop codon can be excluded. To test whether the translation of the downstream
luciferase ORF is due to reinitiation, additional constructs were assayed. The
insertion of an impeding poly(G) stretch decreased this value to 0.0003% (ACT-
pG -LUC in Figure 3.9) corresponding to the luminoscence background activity
of mock-transformed cells (data not shown). In contrast, extending the
intercistronic spacer by duplication of the sequence in construct ACT-119-LUC
resulted in a 31% increase of LUC activity. Thus, the few ribosomes arriving at
AACT
ACTd963
ACTd672
ACTd327
ACTd105
ACTd63
ACTd33
ACTd15
An
63
963
An
AnFLAG-actin170 1157 153
672An
15An
105An
33An
327An
B
AC
T
AC
Td96
3
AC
Td67
2
AC
Td32
7
AC
Td10
5
AC
Td63
AC
Td33
AC
Td15
ACT
PGK1
116±16
ACT/PGK1[%] ± STD
131±20
83±14
42±9
100±9
27±2
19±3
137±11
AACT
ACTd963
ACTd672
ACTd327
ACTd105
ACTd63
ACTd33
ACTd15
An
63
963
An
AnFLAG-actin170 1157 153
672An
15An
105An
33An
327An
ACT
ACTd963
ACTd672
ACTd327
ACTd105
ACTd63
ACTd33
ACTd15
An
63
963
An
AnFLAG-actin170 1157 153
672An
15An
105An
33An
327An
B
AC
T
AC
Td96
3
AC
Td67
2
AC
Td32
7
AC
Td10
5
AC
Td63
AC
Td33
AC
Td15
ACT
PGK1
116±16
ACT/PGK1[%] ± STD
131±20
83±14
42±9
100±9
27±2
19±3
137±11
AC
T
AC
Td96
3
AC
Td67
2
AC
Td32
7
AC
Td10
5
AC
Td63
AC
Td33
AC
Td15
ACT
PGK1
116±16
ACT/PGK1[%] ± STD
131±20
83±14
42±9
100±9
27±2
19±3
137±11
Figure 3.8. 3'-truncation of an actin ORF to 63 nt or shorter triggers decay of themonocistronic mRNA. (A) Map of FLAG-tagged ACT mRNA and 3'-truncation derivatives;numbers indicate lengths in nucleotides. (B) Typical Northern blot of full-length and truncatedFLAG-actin mRNAs, probed for PGK1 and FLAG-tagged ACT mRNAs. The averaged values forrelative LUC mRNA abundance are given under the respective lanes of the Northern blot.
90
3. Results
the downstream ORF in ACT-LUC indeed appear to be reinitiating since both
blocking scanning posttermination ribosomes and facilitating factor recruitment
have been shown to modulate the frequency reinitiation at uORFs (see Section
1.4). However, these experiments are operating at the lower limit of the
sensitivity of luciferase measurement, and due to the enzymatic nature of the
assay values cannot be representative in this range.
3.2.2 Translation of a downstream ORF by reinitiation and leaky scanning can be distinguished using start codon mutants
Having established the first-ORF length range for which the reinitiation
competence of the terminating ribosomes becomes readily measurable, we
investigated the reinitiation-uORF-length relationship in more detail. We started
by generating control constructs in which all the AUGs upstream of LUC were
eliminated by nucleotide substitution (series ∆AUGACTd-LUC, Figure 3.10A,
0.00
100%
0.00
004%
0.00
135%
0.00
119%
0.00
154%
0.00
003%
0.0000%
0.0005%
0.0010%
0.0015%
0.0020%
ACT-LUC
ACT-pG-LUC
ACT-119-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
for protein onlyfor protein and RNA content
corrected forB
AACT-LUC
ACT-pG-LUC
ACT-119-LUC An
119
170 1157 1653AnFLAG-actin luciferase
59 153
An
G18
ACT-LUC
PGK1
AC
T-LU
C
FL'
AC
T-pG
-LU
C
AC
T-11
9-LU
C
LUC
100±5
84±10
LUC/PGK1[%] ± STD
88±7
104±11
C
0.00
100%
0.00
004%
0.00
135%
0.00
119%
0.00
154%
0.00
003%
0.0000%
0.0005%
0.0010%
0.0015%
0.0020%
ACT-LUC
ACT-pG-LUC
ACT-119-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
for protein onlyfor protein and RNA content
corrected for
0.00
100%
0.00
004%
0.00
135%
0.00
119%
0.00
154%
0.00
003%
0.0000%
0.0005%
0.0010%
0.0015%
0.0020%
ACT-LUC
ACT-pG-LUC
ACT-119-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
for protein onlyfor protein and RNA content
corrected forB
AACT-LUC
ACT-pG-LUC
ACT-119-LUC An
119
170 1157 1653AnFLAG-actin luciferase
59 153
An
G18
ACT-LUC
ACT-pG-LUC
ACT-119-LUC An
119
170 1157 1653AnFLAG-actin luciferase
59 153
An
G18
An
119An
119
170 1157 1653AnFLAG-actin luciferase
59 153170 1157 1653AnFLAG-actin luciferase
59 153
An
G18
An
G18
ACT-LUC
PGK1
AC
T-LU
C
FL'
AC
T-pG
-LU
C
AC
T-11
9-LU
C
LUC
100±5
84±10
LUC/PGK1[%] ± STD
88±7
104±11
ACT-LUC
PGK1
AC
T-LU
C
FL'
AC
T-pG
-LU
C
AC
T-11
9-LU
C
LUC
100±5
84±10
LUC/PGK1[%] ± STD
88±7
104±11
C
Figure 3.9. Elements that modulate reinitiation affect the low luciferase activity displayedby the full-length actin-luciferase mRNA. (A) Physical maps of FLAG-tagged actin–luciferase(ACT-LUC) mRNA and derivates with a poly(G) stretch inserted between the ORFs (ACT-pG-LUC) or with the intercistronic spacer extended (ACT-119-LUC). Numbers indicate lengths innucleotides. (B) Relative luciferase activities of constructs, normalised to FL' (not shown) andcorrected for either protein content of the sample only or for both protein content and the relativeLUC mRNA abundance in the sample. (C) Typical steady-state Northern blot of ACT-LUC andmodified transcripts, probed for LUC and PGK1 mRNAs. The averaged values for relative LUCmRNA abundance are given under the respective lanes of the Northern blot.
91
3. Results
Table 2.5). mRNAs with a 5'-UTR derived from the 33 or 63 nucleotide long
ACT ORFs showed 47 and 44% luciferase activity relative to FL', respectively,
after correction for protein and mRNA abundance (Figure 3.10B). However, the
translational activity of construct ∆AUGACTd105-LUC with a longer 5'-UTR was
reduced to 14%. This unexpectedly low translation efficiency was probably due
to the presence of particularly stable secondary structure in this long 5'-UTR
(predicted to have a maximum stability of -75 kcal mol-1, compared to -33 kcal
mol-1 for FL', -45 kcal mol-1 for ∆AUGACTd33-LUC, and -57 kcal mol-1 for
∆AUGACTd63-LUC; according to the mfold algorithm; Zuker et al., 1999). The
reduced translation efficiency of this transcript also correlates with a 2-fold
stabilisation of the ∆AUGACTd105-LUC mRNA. A similar effect was seen upon
IRP1-binding to the leader of construct FL-5'IRE (compare Figure 3.1 and
Figure 3.10). Since we were primarily interested in defining the cut-off point for
reinitiation competence, not all of the construct series included the 15
nucleotide uORF.
As part of a strategy to characterise the contribution of leaky scanning to
initiation on the LUC ORF, the series ACTd∆iAUG-LUC was created, whereby
all AUGs upstream of luciferase with the exception of the actin start codon were
eliminated by nucleotide substitution (Figure 3.10A, Table 2.5). This prevents
initiation on internal codons within the first ORF, for example at any of the three
actin-internal AUGs that are present in the ACTd105-LUC construct (Figure
3.10A, Table 2.5). Moreover, the AUGs that were removed included one that
overlaps in the -1 frame with the UAA stop codon, and which would therefore be
capable of diverting some of the terminating ribosomes into reinitiating at this
site. By normalising to the respective monocistronic construct of the
∆AUGACTd-LUC series we eliminated influences of the different 5'-UTR
sequences upstream of LUC and derived a revised plot of uORF length against
reinitiation (Figure 3.10C).
Consequently, introduction of a 33 nucleotide long (internal AUG-free)
uORF reduced LUC activity to only 54% (after correction for protein and mRNA
abundance), a 63 nt long ACT ORF to 28%, and even ACTd105∆iAUG-LUC,
with an uORF length of 105 nucleotides, still displayed 12% LUC activity. This
compares with 4.3%, 3.1% and 2.0% for the wild type like ACTd-LUC series
comprising actin-internal start codons (Figure 3.10C).
92
3. Results
Some of this initiation at the LUC start codon was expected to be due to
failed recognition of the ACT open reading frame. For increased identification of
Normalised to FL'
0%
10%
20%
30%
40%
50%
60%
15 33 63 105uORF length (in nt)
Rel
ativ
e Lu
cife
rase
Act
ivity
AA
CT-
LUC
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
AU
GA
UG
AC
Td10
5-LU
C
AU
GA
UG
AC
Td63
-LU
C
AU
GA
UG
AC
Td33
-LU
C
∆A
UG
AC
Td10
5-LU
C
∆A
UG
AC
Td63
-LU
C
∆A
UG
AC
Td33
-LU
C
ACT-LUC
PGK1
45±4
84±10
LUC/PGK1[%] ± STD
83±6
42±6
99±4
72±7
53±6
46±7
213±6
140±6
105±11
B
D
170 105 165359 153
ACTd105-LUC
ACTd105∆iAUG-LUC
AUGAUGACTd105-LUC
∆AUGACTd105-LUC * An
* ** An
** ** **ACT AnLUC
* * An
C Normalised to AUGACT d-LUC
0%
10%
20%
30%
40%
50%
60%
33 63 105uORF length (in nt)
Rel
ativ
e Lu
cife
rase
A
ctiv
ity
*
∆AUGACTd-LUC
ACTd∆iAUG-LUCAUGAUGACTd-LUC
ACTd-LUC#
Normalised to FL'
0%
10%
20%
30%
40%
50%
60%
15 33 63 105uORF length (in nt)
Rel
ativ
e Lu
cife
rase
Act
ivity
AA
CT-
LUC
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
AU
GA
UG
AC
Td10
5-LU
C
AU
GA
UG
AC
Td63
-LU
C
AU
GA
UG
AC
Td33
-LU
C
∆A
UG
AC
Td10
5-LU
C
∆A
UG
AC
Td63
-LU
C
∆A
UG
AC
Td33
-LU
C
ACT-LUC
PGK1
45±4
84±10
LUC/PGK1[%] ± STD
83±6
42±6
99±4
72±7
53±6
46±7
213±6
140±6
105±11
AC
T-LU
C
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
AU
GA
UG
AC
Td10
5-LU
C
AU
GA
UG
AC
Td63
-LU
C
AU
GA
UG
AC
Td33
-LU
C
∆A
UG
AC
Td10
5-LU
C
∆A
UG
AC
Td63
-LU
C
∆A
UG
AC
Td33
-LU
C
ACT-LUC
PGK1
AC
T-LU
C
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
AU
GA
UG
AC
Td10
5-LU
C
AU
GA
UG
AC
Td63
-LU
C
AU
GA
UG
AC
Td33
-LU
C
∆A
UG
AC
Td10
5-LU
C
∆A
UG
AC
Td63
-LU
C
∆A
UG
AC
Td33
-LU
C
ACT-LUC
PGK1
45±4
84±10
LUC/PGK1[%] ± STD
83±6
42±6
99±4
72±7
53±6
46±7
213±6
140±6
105±11
B
D
170 105 165359 153
ACTd105-LUC
ACTd105∆iAUG-LUC
AUGAUGACTd105-LUC
∆AUGACTd105-LUC * An
* ** An
** ** **ACT AnLUC
* * An
170 105 165359 153170 105 165359 153
ACTd105-LUC
ACTd105∆iAUG-LUC
AUGAUGACTd105-LUC
∆AUGACTd105-LUC * An* AnAn
* ** An* ** AnAn
** ** **ACT AnLUC** ** **ACT AnAnLUC
* * An* * AnAn
C Normalised to AUGACT d-LUC
0%
10%
20%
30%
40%
50%
60%
33 63 105uORF length (in nt)
Rel
ativ
e Lu
cife
rase
A
ctiv
ity
*
∆AUGACTd-LUC
ACTd∆iAUG-LUCAUGAUGACTd-LUC
ACTd-LUC
∆AUGACTd-LUC
ACTd∆iAUG-LUCAUGAUGACTd-LUC
ACTd-LUC#
Figure 3.10. Manipulation of the FLAG-ACT mini-ORF upstream of LUC. (A) Maps ofACTd105-LUC and further truncated derivatives exemplify four series of constructs. Asterisksindicate the positions of AUGs upstream of the LUC ORF (see Table 2.5 for sequences). uORF-internal start codons are eliminated in the ACTd∆iAUG-LUC series, while a tandem start codonis introduced at the uORF start of this internal AUG-free construct in the AUGAUGACTd-LUCseries. All AUGs upstream of the LUC start codon are mutated in ∆AUGACTd-LUC. (B,C)Relative luciferase activities of AUG-mutated constructs, corrected for both protein content andthe relative LUC mRNA abundance in the sample and normalised to (B) FL' or (C) the respectivemonocistronic ∆AUGACTd-LUC construct. (#) For the ∆AUGACTd-LUC series comprising noupstream AUG, 'uORF length' indicates the size of 5'UTR inserted sequences. (D) Northern blotswere probed for LUC and PGK1 mRNAs; a typical example is shown. The averaged values forrelative ACT-LUC mRNA abundance normalized to FL’ are given under the respective lanes ofthe Northern blots.
93
3. Results
the uORF by scanning ribosomes, a tandem actin start codon was introduced,
yielding the AUGAUGACTd-LUC series to provide the ribosome with a second
opportunity to initiate translation at the uORF in the correct reading frame.
Indeed, luciferase activity of these constructs is reduced by 33% to 48% when
compared to the series ACTd∆iAUG-LUC, which means that the contribution to
LUC initiation from leaky scanning rather than reinitiation has been minimised in
these constructs. The uORF-length / luciferase activity plot obtained with the
AUGAUGACTd-LUC constructs (Figure 3.10C) therefore represents the
relationship that is least distorted by leaky scanning.
However, a striking feature of the plots of uORF length versus relative
luciferase activity is that, although there are variations in quantitative terms,
there is a consistent trend of reduction in reinitiation as length increases (Figure
6C). The construct AUGAUGACTd33-LUC showed 33% luciferase activity,
displaying thereby a high reinitiation rate, whereby an efficiently recognised
uORF of 105 nt almost completely abolishes downstream initiation. This data
provide a strong indication that 35 codons are close to representing the
maximum length of uORF that can support significant reinitiation.
The effectiveness of uORF recognition by ribosomes is also reflected in
mRNA abundance (Figures 3.7C, Figure 3.10D). In both series where initiation
at the ACT start codon or within the uORF was efficient (AUGAUGACTd105-
LUC, ACTd-LUC), the mRNA steady-state levels were generally somewhat
lower than in the constructs with a higher level of 'leaky scanning' or of primary
initiation at the LUC start codon (ACTd∆iAUG-LUC, ∆AUGACTd-LUC).
Recognition and translation of the short uORFs apparently leads to enhanced
mRNA degradation.
3.2.3 Reinitiation competence is affected by elongation rate One potential explanation for the loss of reinitiation competence during the
course of elongation is that one or more of the eIFs remain(s) associated with
the elongating ribosome subsequent to formation of the initial peptide bond, but
then dissociates from the ribosome at a finite rate (McCarthy, 1998). According
to this model, the proportion of ribosomes lacking the eIF(s) would increase with
time, resulting in an apparent ORF length-dependent loss of reinitiation
competence. In order to test this model we carried out experiments in a
temperature-sensitive mutant strain (S. cerevisiae NT33-5) that is defective in
94
3. Results
the Cca1 tRNA nucleotidyltransferase. At the non-permissive temperature for
this mutant, the availability of functional tRNAs in the cell declines, and the rate
of peptide elongation is reduced (Wolfe et al., 1996). At least some mRNA
species are stabilised by this mutation (Peltz et al., 1992).
Measurements with the ACTd∆iAUG-LUC constructs in the mutant
background revealed a general decrease in LUC activity encoded by uORF-
containing mRNAs at the non-permissive temperature (37°C) compared to the
permissive temperature (25°C; Figure 3.11A,B). The data suggest that reducing
the rate of elongation affects the reinitiation rate downstream of an uORF. At
the same time, the relationship between uORF-length and LUC activity is very
different in this strain, whereby the reduction in reinitiation competence as the
uORF increases from 63 to 105 nucleotides is particularly marked. Stalling of
translation elongation also resulted in a more than 10-fold general stabilisation
of LUC mRNA at 37°C, whereas the PGK1 transcript is unstable in heat-
shocked cells (Figure 3.11C). LUC and PGK1 mRNAs are known to respond
differently to inhibition of translation as caused by the shift in temperature in the
Cca1p mutant. Whereas a translationally inhibitory secondary structure in the 5'-
UTR elevates LUC mRNA levels 2-fold (Figure 3.1E), it reduces the half-life of a
PGK1 transcript from ~35 to 3.5 minutes (Muhlrad et al., 1995). Consistently,
LUC mRNA was about 10 to 20-times more stable than PGK1 transcripts when
the cells were shifted from 25°C to 37°C (Figure 3.11C). The increased
sensitivity of reinitiation towards uORF length when aminoacylated tRNAs are
less abundant (compare construct ACTd105∆iAUG-LUC with the dicistronic
constructs harbouring a shorter ORF in Figure 3.11B) is in agreement with a
model whereby the elongating ribosome loses factors necessary for reinitation
(see Section 1.4.3). However, for a reliable comparison, expression of the
tested dicistronic constructs in a reference strain (W303) at the temperatures
assayed (25°C and 37°C) would have to be monitored. In addition, reduced
levels of charged tRNAs result in the activation of the GCN2 / GCN4 pathway
with initiation factor eIF2 becoming less available for translation initiation
(Hinnebusch, 2000). Thus, the observed increased dependence of reinitiation
on uORF length could also stem partly from this indirect effect of Cca1 tRNA
nucleotidyltransferase inactivation. Experiments e.g. in a GCN2 mutant strain
95
3. Results
could help to separate effects of altered eIF2 abundance and uORF length
dependence.
In a study using rabbit reticulocyte lysates, Kozak (2001b) observed a
decrease in reinitiation competence when a pseudoknot that is capable of
slowing down elongating ribosomes was inserted into a 54 nucleotide uORF.
This parallels the effect we see in the mutant strain where elongation is
restricted due to a decrease in the level of aminoacylated tRNAs. Both our
study and that of Kozak are consistent with the idea that the kinetics of
corrected for
87%
106%
90%
57%
100%
63%
84%
77%
19%100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
37
ºC /
25ºC
protein content onlyprotein and RNA content
∆∆ ∆ ∆
81%
86%
64%2%100%
51%
73%
49%
0.4%
100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
corr
ecte
d fo
r pro
tein
and
RN
A co
nten
t
25ºC37ºC
∆∆ ∆ ∆
A
B C
FL"
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
FL”
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
LUCACT-LUC
PGK1
25ºC 37ºC
51±7
100±8
LUC/PGK1[%] ± STD
90±14
1224±174
129±17
81±5
1869±228
1850±280
1391±199
1376±213
corrected for
87%
106%
90%
57%
100%
63%
84%
77%
19%100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
37
ºC /
25ºC
protein content onlyprotein and RNA content
∆∆ ∆ ∆
87%
106%
90%
57%
100%
63%
84%
77%
19%100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
37
ºC /
25ºC
protein content onlyprotein and RNA content
∆∆ ∆ ∆
81%
86%
64%2%100%
51%
73%
49%
0.4%
100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
corr
ecte
d fo
r pro
tein
and
RN
A co
nten
t
25ºC37ºC
∆∆ ∆ ∆
81%
86%
64%2%100%
51%
73%
49%
0.4%
100%
0%
25%
50%
75%
100%
125%
FL"
ACTd105
iAUG-L
UC
ACTd63
iAUG-L
UC
ACTd33
iAUG-L
UC
ACTd15
iAUG-L
UC
Rel
ativ
e Lu
cife
rase
Act
ivity
corr
ecte
d fo
r pro
tein
and
RN
A co
nten
t
25ºC37ºC
∆∆ ∆ ∆
A
B C
FL"
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
FL”
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
LUCACT-LUC
PGK1
25ºC 37ºC
51±7
100±8
LUC/PGK1[%] ± STD
90±14
1224±174
129±17
81±5
1869±228
1850±280
1391±199
1376±213
FL"
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
FL”
AC
Td10
5∆iA
UG
-LU
C
AC
Td63
∆iA
UG
-LU
C
AC
Td33
∆iA
UG
-LU
C
AC
Td15
∆iA
UG
-LU
C
LUCACT-LUC
PGK1
25ºC 37ºC
51±7
100±8
LUC/PGK1[%] ± STD
90±14
1224±174
129±17
81±5
1869±228
1850±280
1391±199
1376±213
Figure 3.11. Inhibition of translation elongation affects expression of a downstreamluciferase ORF in a uORF-size dependent manner. (A) Luciferase activities of cell lysatesextracted from strain NT33-5 carrying a heat-sensitive allele of the ATP(CTP):tRNAnucleotidyltransferase-encoding gene CCA1 grown for 4 hours at either 25°C or at the non-permissive temperature of 37°C. The values are normalised to FL" (which corresponds to FL butcarries a URA3 marker) for each temperature and corrected for both protein and RNAconcentrations in the sample. (B) Ratios of luciferase activities of cells grown at 37°C or 25°C,corrected for either protein content of the sample only or for both protein content and the relativeLUC mRNA abundance in the sample. (C) Northern blots were probed for LUC and PGK1mRNAs, the averaged values for relative LUC mRNA abundance are given under the respectivelanes of the Northern blot.
96
3. Results
elongation influence reinitiation competence. This can be explained by a model
in which one or more of the eIFs does not immediately leave the ribosome
subsequent to initiation of peptide synthesis, thus maintaining the ribosome in a
reinitiation-competent state. Assuming a finite (relatively low) rate of release of
the eIF(s), reinitiation competence will be progressively lost as a function of time
and / or progression through the uORF. However, the data we have obtained
with the cca1 mutant indicate that the rate of loss of the eIF(s) in such a model
would not simply be a straightforward function of time, but rather also influenced
by other factors. Consequently, the elongation rate influences the maximum
length of uORF that can allow ribosomes to retain their reinitiation competence.
3.2.4 Premature translation termination by either ORF truncation or stop codon insertion affects mRNA stability differently
3.2.4.1 The presence of premature termination codons but not 3'-truncation of the ORF trigger nonsense-mediated decay
It is known that translation termination can trigger accelerated decay of mRNA
(McCarthy, 1998; Jacobson & Peltz, 2000). The strongest effect is seen when
premature termination codons (PTCs) in a reading frame cause nonsense-
mediated decay (Jacobson & Peltz, 2000; Maquat, 2000, Section 1.3.2).
Previous studies of nonsense-mediated control in yeast have focused on
premature termination within an otherwise intact gene (Gonzalez et al., 2001).
Yet, the 3' truncation of the actin ORF in the ACTd series results in a special
kind of premature termination, whereby the length and the composition of the 3'-
UTR remains unaffected (Figure 3.8). For comparison, therefore, we generated
a set of premature termination codon (PTC) constructs by introducing a UAA
stop codon at several positions within the actin ORF. Translation on these
mRNA is terminated 963, 672, 327 or 63 nt downstream of the start codon, and
the remaining actin ORF – together with the FL' inherent trailer – acts as an 3'-
UTR (Figure 3.12). In contrast to the ACTd-LUC 3'-UTRs, this additional
sequence extends the distance between the stop codon and the poly(A) tail by
about 200 – 1000 nt, depending on the site of the PTC. Moreover, the
remainder of the actin ORF also comprises several start and in-frame stop
codons, creating thereby the possibility for downstream ribosomes to undergo
cycles of translation at these ORFs of varying lengths.
97
3. Results
G
ACT-LUC
ACTptc963-LUC
ACTptc672-LUC
ACTptc327-LUC
ACTptc63-LUC
AnFLAG-actin luciferase170 1157 165359 153
327 PTCAn
An
963 PTC
672An
PTC
63AnFLAG-actin
PTC
ACT
ACTptc963
ACTptc672
ACTptc327
ACTptc63
C
E
B
ACT
AC
Tptc
963
AC
Tptc
672
AC
Tptc
327
AC
Tptc
63
AC
T
PGK1
100±5
14±7
11±9
12±10
110±12
ACT/PGK1[%] ± STD
ACT-LUC
PGK1
FL'
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
LUC
100±12
119±6
LUC/PGK1[%] ± STD
56±4
65±6
76±6
80±2
F
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
A
0.00
005%
0.00
011%
0.00
006%
0.00
013%
0.00
100%
0.00
119%
0.00
008%
0.00
023%
0.00
009%
0.00
024%
0.0000%
0.0005%
0.0010%
0.0015%
ACT-LUC
ACTptc9
63-LUC
ACTptc67
2-LUC
ACTptc32
7-LUC
ACTptc63
-LUC
Rel
ativ
e Lu
cife
rase
Act
ivity protein content only
protein and RNA content
corrected forD
100%
110%
14%
11%
12%
100%
65%
80%
56%
76%
0%
25%
50%
75%
100%
125%
ACT
ACTptc96
3
ACTptc67
2
ACTptc32
7
ACTptc63
Rel
ativ
e m
RN
A a
bund
ance
LUC
/PG
K1
monocistronicdicistronic (+LUC)
An
63
963
AnFLAG-actin
AnFLAG-actin170 1157
672An
327
PTC
PTC
PTC
PTCAn
153
G
ACT-LUC
ACTptc963-LUC
ACTptc672-LUC
ACTptc327-LUC
ACTptc63-LUC
AnFLAG-actin luciferase170 1157 165359 153
327 PTCAn
An
963 PTC
672An
PTC
63AnFLAG-actin
PTC
ACT-LUC
ACTptc963-LUC
ACTptc672-LUC
ACTptc327-LUC
ACTptc63-LUC
AnFLAG-actin luciferase170 1157 165359 153
327 PTCAn
An
963 PTC
672An
PTC
63AnFLAG-actin
PTC
AnFLAG-actin luciferase170 1157 165359 153
327 PTCAn
An
963 PTC
672An
PTC
63AnFLAG-actin
PTC
ACT
ACTptc963
ACTptc672
ACTptc327
ACTptc63
C
E
B
ACT
AC
Tptc
963
AC
Tptc
672
AC
Tptc
327
AC
Tptc
63
AC
T
PGK1
100±5
14±7
11±9
12±10
110±12
ACT/PGK1[%] ± STD
ACT
AC
Tptc
963
AC
Tptc
672
AC
Tptc
327
AC
Tptc
63
AC
T
PGK1
100±5
14±7
11±9
12±10
110±12
ACT/PGK1[%] ± STD
ACT-LUC
PGK1
FL'
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
LUC
100±12
119±6
LUC/PGK1[%] ± STD
56±4
65±6
76±6
80±2
ACT-LUC
PGK1
FL'
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
LUC
100±12
119±6
LUC/PGK1[%] ± STD
56±4
65±6
76±6
80±2
F
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
32.5
16.5
47.5
25.0
Act1p
kDa
32.5
16.5
47.5
25.0
Act1p
kDa
AC
T-LU
C
AC
Tptc
963-
LUC
AC
Tptc
672-
LUC
AC
Tptc
327-
LUC
AC
Tptc
63-L
UC
A
0.00
005%
0.00
011%
0.00
006%
0.00
013%
0.00
100%
0.00
119%
0.00
008%
0.00
023%
0.00
009%
0.00
024%
0.0000%
0.0005%
0.0010%
0.0015%
ACT-LUC
ACTptc9
63-LUC
ACTptc67
2-LUC
ACTptc32
7-LUC
ACTptc63
-LUC
Rel
ativ
e Lu
cife
rase
Act
ivity protein content only
protein and RNA content
corrected for
0.00
005%
0.00
011%
0.00
006%
0.00
013%
0.00
100%
0.00
119%
0.00
008%
0.00
023%
0.00
009%
0.00
024%
0.0000%
0.0005%
0.0010%
0.0015%
ACT-LUC
ACTptc9
63-LUC
ACTptc67
2-LUC
ACTptc32
7-LUC
ACTptc63
-LUC
Rel
ativ
e Lu
cife
rase
Act
ivity protein content only
protein and RNA content
corrected forD
100%
110%
14%
11%
12%
100%
65%
80%
56%
76%
0%
25%
50%
75%
100%
125%
ACT
ACTptc96
3
ACTptc67
2
ACTptc32
7
ACTptc63
Rel
ativ
e m
RN
A a
bund
ance
LUC
/PG
K1
monocistronicdicistronic (+LUC)
An
63
963
AnFLAG-actin
AnFLAG-actin170 1157
672An
327
PTC
PTC
PTC
PTCAn
153
Figure 3.12. Impact of a premature stop codon on the translation and abundance ofmonocistronic ACT and bicistronic the ACT-LUC mRNA. (A) Maps of FLAG-tagged ACT mRNAand mutants with a premature termination codon (PTC). Numbers indicate lengths in nucleotides. (B) Typicalsteady-state Northern blot of FLAG-actin and PTC-mutants, probed for FLAG-ACT and PGK1 mRNAs. Theaveraged values for relative FLAG-ACT mRNA abundance are given under the respective lanes of the Northernblot. (C) Maps of FLAG-tagged ACT-LUC mRNAs with a premature termination codon (PTC) was inserted atseveral positions. The remaining (white-boxed) ACT ORF forms part of the intercistronic spacer. Numbers indicatelengths in nucleotides. (D) Relative luciferase activities of constructs, normalised to FL' (not shown) and correctedfor either protein content of the sample only or for both protein and ACT-LUC RNA concentrations. (E) Western blotshowing expression of the actin ORF using a FLAG-antibody. The full-length actin protein is indicated. (F) TypicalNorthern blot of FL' and bicistronic constructs, probed for LUC and PGK1 mRNAs. The averaged values for relativemRNA abundance are given under each lane. (G) Comparison, for each construct, of the relative ACT mRNAabundance with and without the downstream LUC ORF.
98
3. Results
These monocistronic reference mRNAs showed significant mRNA
destabilisation when a PTC was located in the first two thirds of the actin ORF.
Whereas PTC insertion in construct ACTptc963 with 83% of the full-length
FLAG-actin ORF being translated had no obvious effect on mRNA stability,
reducing the translated region to 58% (ACTptc672) or less (ACTptc327,
ACTptc63) resulted in NMD-like accelerated decay (Figure 3.12B). However, to
verify degradation via the NMD pathway, the stability of these constructs would
have to be assessed in a genetic background deficient for components of this
pathway (e.g. the Upf proteins).
3.2.4.2 PTC-containing mRNAs are stabilised by a full-length downstream ORF that is not translated
To test for reinitiation downstream of PTCs, a luciferase cassette was inserted
downstream of the wild type actin stop codon of ACTptc constructs, yielding the
ACTptc-LUC constructs. The resulting bicistronic constructs differed from
ACTd-LUC in that the spacer also comprises the actin sequence downstream of
the PTC (compare Figures 3.7A; 3.11C). In this case, reinitiation at the LUC
ORF remained highly inefficient, even with the very shortest actin-derived first
ORF. Intercistronic AUG codons from the original actin ORF lie downstream of
the newly created PTCs, and these are all capable of 'capturing' reinitiation-
competent ribosomal subunits. Initiation and translation of the actin ORF was
efficient as shown by detection of the full-length actin protein in an anti-FLAG
Western blot (Figure 3.12E).
However, introduction of the downstream LUC ORF markedly stabilised
the decay-susceptible PTC-containing mRNAs (as shown in the comparison
provided by Figure 3.12G). Since the dicistronic constructs display no significant
luciferase activity, mRNA stabilisation appears not to be linked to translation of
the downstream ORF. Moreover, this effect seems to be restricted to mRNAs
susceptible to NMD, since mRNAs with truncated actin ORFs were not
stabilised to the same degree by insertion of a LUC ORF in the 3'-UTR
(compare Figures 3.7C, 3.8B and Figures 3.12B, 3.12F, 3.12G). In summary,
the relative levels of mRNA abundances and LUC translation rates for the
truncated bicistronic and monocistronic transcripts showed no evident
relationship to the mRNA degradation and reinitiation situation at PTC-carrying
transcripts. Therefore, the situation of the terminating ribosome must be
99
3. Results
different at these two kinds of shortened ORFs, affecting its subsequent
posttermination fate (reinitiation) and the effect on mRNA stability.
100
4. Discussion
4. DISCUSSION
4.1 Accessibility of the 3'-UTR is not a prerequisite for posttermination ribosome recycling
4.1.1 Differences between 5'-UTR and 3'-UTR directed blockage In this study, we show that imposing a block to ribosomal movement along the
3'-UTR of an in vivo expressed reporter mRNA did have a limited and position-
dependent effect on translational efficiency. When compared to the 95%
reduction obtained by restricting access to the 5'-UTR, IRP1-binding 24 nt or 39
nt downstream of the luciferase stop codon decreased translation only by about
27%. When the IRP1 binding site was close enough to the stop codon for the
terminating ribosomes to interfere with the complex, no effect on LUC activity
was apparent, suggesting that the terminating ribosome has sufficient energy to
destabilise IRP1-binding to IRE (construct FL-15-IRE in Figure 3.1).
Alternatively, the stop codon 3'-proximal structure might even aid in recognition
of the termination site as it was observed for the identification of start codons in
a suboptimal context by 40S subunits (Kozak, 1991). IRP1-association with the
3'-UTR at a position close to the poly(A) tail resulted in a slight (~25%) increase
in mRNA stability and a ~50% decrease in translational efficiency (FL-141-IRE).
This more pronounced effect could be due to the close vicinity of the poly(A) tail
to the IRP1-binding site and because of the presence of tandem copies of IRE,
presumably resulting in a more stable IRP1-blockage.
Niepel and colleagues (1999) observed a ~50% decrease in translational
efficiency when they placed a strong stem loop close adjacent to the stop
codon, but their experiments differed from ours since they programmed yeast
spheroblasts and used an inherent stable structure that is not readily
destabilised unlike IRE that is not bound by IRP1 (see also Section 3.1.2). They
also observed a gradual loss of this inhibitory effect when inserting a 20 nt or
100 nt long spacers between the translation termination and the polyadenylation
site. The monitored reduction by 33% and 22%, respectively, was only
detectable in yeast spheroblasts, whereas in mammalian or plant cell-free and
protoblast systems movement of the structure downstream of the stop codon
relieved the repression completely. This indicates not only an enhanced
101
4. Discussion
sensitivity of yeast cells towards secondary structures in the 3'-UTR (as already
known for the 5'-UTR), but also confirms the position-dependence of the
translational repression by the IRE – IRP1 system.
The observed IRE – IRP1 induced reduction in reporter gene activity was
exclusively caused by a decrease in translational efficiency of the LUC mRNA,
since (1) the mRNA was not destabilised, (2) the poly(A) tail length and
polyadenylation sites were not affected, and (3) the LUC mRNA distribution
among the polysomal profiles correlated with the decrease in translation. In the
system presented, the IRP1-IRE complex almost completely abolished gene
expression when located in the 5'-UTR, most likely due to an inhibition of 43S-
cap association and scanning of the leader (Koloteva et al., 1997). However,
the 3'-UTR located blockage imposed a comparably low effect on LUC
translation, indicating thereby that the accessibility of the sequences
downstream of the termination site to scanning ribosomes is only of minor
importance for the translational efficiency. Assuming that Pab1p-contributed
increase in translation initiation frequency is partly due to 'channelling' and
recycling of ribosomes or subunits, it can be deduced that this does not occur
via 3'-UTR scanning, contrasting with the reinitiation mechanism that relies on
scanning downstream of termination. This perception is supported by the finding
that mutating the LUC stop codon context as to facilitate ribosomal release at
the termination site and to prevent reinitiation did not affect translational
efficiency of the mRNA. Moreover, repression by IRP-binding to the 3'-UTR did
not rely on the existence of an efficient 5' – 3' interactions as revealed by
assaying construct FL-39-IRE in a yeast strain expressing an eIF4GI mutant
defective for Pab1p binding. Similarly, Niepel and colleagues (1999) observed
no interruption of the poly(A) – cap synergy by insertion of 3'-UTR secondary
structures in yeast spheroblasts.
However, in the in vivo system assayed the yeast cells were allowed to
grow in rich medium at a favourable temperature (30°C). In this optimal
environment it cannot be excluded that a reduction in translational activity
caused by the 3'-UTR blockage was compensated by the enhanced utilisation
of an alternative pathway. For example, blocking the intramolecular recycling
pathway could cause an enhanced de novo recruitment of ribosomal subunits
from the cellular pool. In further experiments, it would be interesting to monitor
102
4. Discussion
the effect of 3'-UTR secondary structures in cells grown under stress conditions
or in the stat phase with enhanced competition between mRNAs for recruitment
of ribosomes.
4.1.2 Translation might be affected by a 3'UTR-conferred mRNP modification
The data suggest that modification of the 3'-UTR affects the translatability of a
cellular mRNA by virtue of the fact that it changes mRNP structure, rather than
because it blocks the passage of ribosomes through the 3'-UTR. This would
also explain the 2-fold reduction in translation when placing the IRE-IRP1
complex further downstream, close to the poly(A) tail. This could not be
explained by blocking a processive event such as the scanning-like association
of ribosomes with the 3'-UTR but indicates a more indirect, position-dependent
effect like changes of the mRNP structure. In the yeast cell-free system, the
normal mRNP structure is already partially disrupted, and this explains why
changes in the 3'-UTR of this kind have no detectable effect. This does not
exclude that mRNAs are circularised in vitro (as shown by Wells et al., 1998).
The presence of a poly(A) tail stimulates translation of mRNAs regardless of its
conformation caused by IRP1-binding (Figure 3.6C,D) or by secondary
structures (Niepel et al., 1999). However, at least the quantitative principles of
translational control on a template that is located within a non-physiological
mRNP structure will be different to those applying to normal cellular polysomes.
In this context, the results of Borman and colleagues (58) are worthy of note.
They observed that, in rabbit reticulocyte lysate, exogenous poly(A) stimulates
translation of capped, nonadenylated mRNA to the same degree as the
presence of a poly(A) tail at the 3'-end of the mRNA. This underlines the fact
that mRNP functions differently in a cell-free system, at least to the extent that
internal 5' – 3' interactions are not required for translation efficiency to be
maximised.
Similar to the proposed reliance on mRNP domain organisation for mRNA
surveillance (Hilleren & Parker, 1999), translation depends on mRNP domains
such as those defined by the cap-binding complex, the factors associated with
translation initiation and termination sites, and the 3'-UTR / poly(A) attached
proteins. Although not directly interfering with ribosomal scanning, downstream
structures could therefore disturb this proper organisation and e.g. disrupt
103
4. Discussion
termination site – poly(A) interactions granted by the eRF3 – Pab1p association.
Likewise, Curatola et al. (1995) showed that a secondary structure at any
position between the stop codon and a 3'-UTR located ARE sequence can
prevent ARE-induced degradation in HeLA cell extracts. They suggest this
might due blocked propagation of conformational or compositional changes to
the 3'-UTR RNP that might result from translation of the 3'-terminus of the
coding region rather than from entrance of translation-linked factors into the 3'-
UTR (i.e. posttermination scanning). Interestingly, IRE-insertion and IRP1-
binding to the 3'-UTR did not result in destabilisation of LUC mRNA. This
indicates that the induced mRNP distortion only affected translation, but not any
of the mRNA stability determinants such as the length of the poly(A) tail, its
association with Pab1p or the eIF4G – Pab1p interaction.
A modified 'circular loop' model of mRNA
In context of the mRNP model, the interaction between the termination site and
nAAAA
ORF
60S4E
eIF4G
Pab1p eR
F3eR
F1
ribosomal release
initiation
termination
elongation
scanning
40S recruit-
ment
'chan
nellin
g'
40S40S
nAAAA
ORF
60S4E
eIF4G
Pab1p eR
F3eR
F1
ribosomal release
initiation
termination
elongation
scanning
40S recruit-
ment
'chan
nellin
g'
40S40S40S40S
Figure 4.1. Channelling is promoted by mRNP structure. The protein bridge Pab1-eIF4G-eIF4E canmediate interactions between the 5’ and 3’-ends of mRNA. The interaction between eRF3 and Pab1 mayalso link the termination site with the poly(A) tail. According to our data, while such structures within thecellular mRNP promote recycling of ribosomes, this does not require continuous scanning along the 3'-UTR. Instead, juxtaposition of the sites of termination and initiation facilitates the process. This contrastswith the process of reinitiation, which is only found to occur at a significant efficiency downstream of uORFsof up to 35 codons in length. Reinitiation is strictly dependent upon scanning between the site of terminationand the downstream start site. The dotted hairpin alludes to the positioning of structural elements in the 3'-UTR that were used in this work.
104
4. Discussion
the poly(A) tail would cause the 3'-UTR to loop out of an already circular mRNA,
as proposed by Uchida and colleagues (2002). Channelling of ribosomes or
subunits would occur not via the 3'-UTR – Pab1p – eIF4F – cap interaction but
due to the proximity of termination site and 5'-UTR afforded by the eRF3 –
Pab1p – eIF4F – cap bridge (Figure 4.1). As a consequence, this would make
properties such as secondary structures in the 3'-UTR ineffective since they
would not block a 'bottleneck' step such as 3'-UTR scanning. Interestingly,
eRF3 remains associated with 40S subunits after termination (Didichenko et al.,
1991; Eustice et al., 1986), enabling an uninterrupted guidance of the subunit
via Pab1p to the 5'-end of a circular mRNA without requiring involvement of the
3'-UTR sequence.
4.1.3 Recycling is unlikely to be reinitiation-like since the ribosomal reinitiation potential depends on the ORF length
In this study, we also consequently investigated the dependence of ribosomal
reinitiation capability on ORF-length. We found that the reinitiation of translation
at a downstream ORF is practically zero when the ribosome has translated a
full-length wild type like open reading frame of about 1.2 kb. Reinitiation was
only readily detectable with the ORF 3'-truncted to 35 codons (105 nt) or less.
This is in agreement with a predicted cut-off length of 55 codons (168 nt) on
viral mRNA (Luukkonnen et al., 1995) and a gradual decrease in reinitiation by
extending the uORF up to 33 codons (102 nt) in a rabbit reticulocyte cell-free
lysate (Kozak, 2001). Moreover, experiments in a cca1 mutant with reduced
aminoacylated tRNA levels at a non-permissive temperature suggest a model
whereby the length of the ORF in relationship with the elongation cycle kinetics
determines the stage in elongation at which the ribosome is rendered unable to
promote posttermination reinitiation. Introduction of short uORFs of varying
length resulted in a more pronounced inhibition of downstream initiation when
translation elongation was slowed down by above measure (Figure 3.11).
Hence, the properties of ribosomes terminating at a short uORF and at a long
ORF differ fundamentally, most likely in their association with factors required
for reinitiation such as eIF3 and GTP-charged eIF2. In agreement with the
limited effect of 3'-UTR structures on translational efficiency, this finding
provides additional evidence that intramolecular ribosome recycling does not
rely on a posttermination scanning and reinitiation-like step. In this context, it
105
4. Discussion
will be of great interest to determine at which step in recycling ribosomes
reacquire the ability to initiate translation. Our results indicate that in contrast to
reinitiation after a short uORF, this recruitment appears not to be facilitated by a
posttermination scanning step.
4.2 The relationship between ORF length, nonsense-mediated decay and reinitiation
4.2.1 PTC-insertion but not ORF 3'-truncation results in accelerated mRNA decay
To further investigate the role of ORF length and the 3'-UTR on translation and
mRNA degradation, we assessed the stability of two groups of mRNAs: The first
comprised a single, progressively 3'-truncated ORF with a 3'-UTR of constant
length (Figure 3.7, Figure 3.8). The second had the ORF interrupted by a
premature termination codon (PTC), thus extending the 3'-UTR accordingly.
Interestingly, only insertion of the PTC, but not 3'-truncation resulted in
accelerated decay. Rapid mRNA degradation due to the presence of a
premature stop codon has been shown for other mRNAs to occur via the NMD
pathway at comparable rates (see Section 1.3.2.2 for details). Abolishing this
pathway, e.g. in a mutant background deficient for one of the Upf proteins could
verify the mechanism of mRNA degradation for both the PTC-containing and
the truncated ORFs. According to the model by Peltz and colleagues (1993),
the observed difference in the degradation rates would be due to the lack of a
downstream destabilising sequence element (DSE) in the truncated constructs.
In agreement, the only construct that is not destabilised has the PTC located
about 200 nt upstream of the regular termination site, probably deficient for a
downstream DSE. However, this mRNA also comprises only a single
downstream ORF whereas the other PTC-carrying transcripts contain several
downstream AUGs and inframe stop codons in the extended sequence between
stop codon and poly(A) tail. In an alternative model, it might therefore be
possible for ribosomes terminating at the prematurely encountered stop codons
to resume scanning and to reinitiate. Consequently, the numerous downstream
ORFs all terminate in a distance from the 3'-UTR and the resulting unfavourable
mRNP environment might be responsible for triggering the accelerated mRNA
decay. Conversely, the truncated constructs all have a 3'-UTR with a constant
106
4. Discussion
length which might help to stabilise these transcripts. This might be a feature
characteristic for S. cerevisiae since in this organism 3'-UTRs are strikingly
homogenous in length (see also Section 1.3.2.3).
4.2.2 Insertion of an untranslated downstream ORF stabilises PTC-carrying mRNAs
The introduction of a PTC into the first two thirds of the actin ORF increases the
effective length of the 3’UTR, resulting in marked destabilisation of the mRNA.
This is entirely consistent with previous work on the effects of PTCs on the
stability of other yeast messages (including PGK1 and URA1; reviewed in
McCarthy, 1998, and Jacobson & Peltz, 1996). However, this effect was
relieved to a certain degree when a full-length ORF was inserted downstream of
the PTC-containing ORF. We found that the downstream LUC ORF is not
translated in these constructs but observed an unexpected 4 to 7-fold
stabilisation of the mRNAs upon downstream ORF insertion (Figure 3.12G). No
such stabilisation was found when the luciferase ORF was appended to
truncated actin ORFs, but there was a general tendency to destabilisation when
the actin ORF was truncated to 105 nt or less (compare Figure 3.7 and Figure
3.8). This corresponds to a progressive replacement of the stable actin mRNA
sequence by the less stable LUC ORF.
Another earlier study has shown that extension of the 3'-UTR of the CUP1
ORF resulted in NMD-like destabilisation (Muhlrad & Parker, 1999). Although
addition of the (untranslated) LUC ORF practically extends the 3'-UTR by 1,700
nt, our findings differ from this and point to a general stabilising function of the
added sequence that is also not correlated to its relative position to the
(premature) termination codon. This indicates that several parts of the LUC
reading frame might contribute to determine mRNA stability, probably by
defining an overall mRNP structure different from the PTC-mRNA with a single
ORF.
4.2.3 Modulation of leaky scanning and mRNA stability by the efficiency of uORF recognition
The presence of an upstream ORF derived from the wild type, FLAG-tagged
yeast actin sequence in the leader of a luciferase reporter mRNA resulted in a
high repression of downstream translation, even when the uORF was truncated
to 15 residues (4 amino acids). Since this restriction was upheld in constructs
107
4. Discussion
ranging from 15 to 105 nt, a 'ribosome stalling' effect of the sequence 5' of the
uORF stop codon (that varied in these constructs) can be ruled out.
To illustrate the uORF initiation and termination events at construct
ACTd105-LUC, Figure 4.2 depicts all 5’-UTR located uORFs arising by the
utilisation of the ATGs upstream of the LUC ORF. At this wild type sequence,
leaky scanning across the first ATG will result in ribosomes translating the
second uORF. Translation of this uORF2 is shifted in frame (by +1) with respect
to uORF1 and terminates 46 nt upstream of the main LUC ORF. Furthermore,
all other uORFs terminate in this region and all of them are shorter in length
than uORF1. Therefore, ribosomes having translated any part of the leader can
be expected to have comparable reinitiation capabilities. Moreover, due to
overlapping ORFs it can also be excluded that a particular ribosomes translates
more than one uORF. Therefore, the observed low reinitiation rate for this
construct cannot be the result of leaky scanning or multiple translation events
upstream of the main LUC ORF. This is also true for the constructs with a
truncated uORF. However, the highly restrictive control on reinitiation was vastly
relieved (10-fold with a uORF of 33 nt) when the native uORF-internal start
codons were mutated (Figure 3.10).
To investigate the contribution of ribosomes arriving at the luciferase start
codon via 'leaky scanning', we inserted a double uORF start codon in these
internal AUG-free mutants. Indeed, downstream translation was reduced
proportionally indicating to be more reliant on reinitiation in these constructs. To
explain the even more stringent effect of wild type uORF sequences with
inherent uAUGs and uORFs, a model might be useful that entails the negative
effects of initiating and terminating ribosomes (and subunits) on scanning and
elongation (Figure 4.3). Whereas in the (artificial) constructs with only a single
or double (inframe) uORF AUG, scanning, elongation and (re)scanning occurs
________________________________________________main uORF________________________________________________----spacer--->>ATGGATTACAAGGACGACGATGACAAGACTATGGATTCTGAGGTTGCTGCTTTGGTTATTGATAACGGTTCTGGTATGTGTAAAGCCGGTTTTGCCGGTGACTAATGTAGAATTCTAGAA________________________________________________main uORF________________________________________________----spacer--->>ATGGATTACAAGGACGACGATGACAAGACTATGGATTCTGAGGTTGCTGCTTTGGTTATTGATAACGGTTCTGGTATGTGTAAAGCCGGTTTTGCCGGTGACTAATGTAGAATTCTAGAA
Figure 4.2. 5'-UTR sequence of ACTd105-LUC and representation of ORFs starting at allpossible upstream ATGs. The 5' to 3' sequence of the 105 nt long uORF is representedtogether with the first nucleotides of the 59 nt long intercistronic spacer. Boxes indicate thesequences covered by ORFs starting at all possible uATGs with gray boxes indicating ORFs ina different reading frame. Note that all uORFs extend to a maxiumum of 13 nt into the spacer,terminating thereby at least 46 nt upstream of the (not depicted) downstream LUC ORF. ATGsare in bold, termination codons are italic.
108
4. Discussion
in an ordered manner, the situation at a uORF with internal initiation and
termination sites might be more complex. Ribosomes that have arrived at these
sites by leaky scanning or translation of (internal) uORFs could potentially
hinder the movement of scanning or elongating ribosomes, thus slowing them
down or causing them to abort their association with the mRNA, resulting in
ribosomal release. The effects of these internal ORF would therefore be greater
than their contribution to the reinitiation cycles on these transcripts. Ribosomes
stalled at termination sites of uORFs only translated by 10% of the subunits
have been found to efficiently block movement of subsequent ribosomes and
subsequent (re)initiation at the main ORF start codon (see also Section 1.4.4).
Similarly, the described system will only work within a leader where the (main)
uORF is not recognised by all scanning subunits. This is true for the uORF start
codon context used in this study, as revealed by the decrease in downstream
initiation upon creation insertion of a second uORF start codon Interestingly,
GCN4, YAP1, ermC, cm1A and potentially other natural uORFs do not
*
* *
*
* **
** *
∆AUGACTd33-LUC
ACTd33∆iAUG-LUC
AUGAUGACTd33-LUC
ACTd33-LUC (wild type)
LUCA
B
C
D
100%
54%
33%
4%
LUC activity
scanning
* = AUG
ribosome release
uORF
translation
posttermination scanning
*
* *
*
* **
** *
∆AUGACTd33-LUC
ACTd33∆iAUG-LUC
AUGAUGACTd33-LUC
ACTd33-LUC (wild type)
LUCA
B
C
D
100%
54%
33%
4%
LUC activity
scanning
* = AUG
ribosome release
uORF
translation
posttermination scanning
Figure 4.3. A ribosome flow model for the inhibition of downstream (re)initiation byuORFs comprising uAUGs. The fates of (40S) ribosomal subunits entering the 5’-ends of AUG-mutated bicistronic ACTd33-LUC mRNAs are depicted; the thickness of the respectivearrows corresponds to a semi-quantitative representation. The LUC activity values normalised to ∆AUGACTd33-LUC are taken from Figure 3.10B. (A) Virtually all scanning 40S subunits start translation at the main ORF on an uORF-free mRNA. (B) A single uAUG with an inframe stop codon diverts most of the scanning subunits to translation of the uORF, a small part will reach the downstream AUG via ‘leaky scanning’. Having translated the uORF, part of the ribosomeswill be released from the mRNA, some subunits will resume scanning and reinitiate translation.(C) A tandem start codon enhances recognition of the uORF; initiation at the downstream ORF will drop because of significantly reduced ‘leaky scanning’ and an elevated release of ribosomesat the uORF stop codon. (D) On the ‘wild-type’ uORF of construct ACTd105-LUC 40S subunits cannot only initiate at 3 uAUGs, but scanning, elongating and terminating ribosomes arerepeatedly blocked by ribosomes at translation initiation and termination sites. This blockagemultiplies the negative effects of all uORFs, resulting in only a small fraction of ribosomesreaching the LUC main ORF.
109
4. Discussion
comprise internal AUGs, and in cases of overlapping uORF such as in YAP2
and connexin-41 these appear to be used deliberately as a means of regulation.
The efficiency of uORF recognition and initiation upstream of the LUC
ORF is also related closely to the stability of the uORF-containing mRNA
(Figure 3.10). Effective translation of the upstream ORF and lack of proper
termination results in accelerated mRNA decay, whereas translation of
luciferase due to leaky scanning or absence of uORFs stabilises the mRNA.
4.3 Future perspectives The results of this study indicate that posttermination scanning of the 3'-UTR
contributes only minimally to ribosome recycling within the mRNA. Recent work
suggests that the sites of termination and initiation are held in close proximity
via the interaction chain eRF3 – Pab1p – eIF4F, thus providing a different
model for ribosomal 'channelling'. Preliminary experiments by Uchida et al.
(2002) indicate that eRF3 – Pab1p binding is involved only in subsequent
rounds of translation, and not in the de novo formation of the 80S complex. In
order to test this type of model further, it will be a major challenge to
demonstrate the existence of 'channelling' ribosomes of this kind and to
determine whether the whole 80S ribosome or its subunits are guided back to
the 5'-end. Genetically, it might be possible to dissect the contribution of Pab1p
and eRF3 to ribosome recycling by fusing the eIF4G-binding site of Pab1p to
eRF3 in a mutant background with the eIF4G – Pab1p interaction disrupted
(e.g. strain YAS1947).
The second major fate for the posttermination ribosome (in addition to
recycling via release or 'channelling') is reinitiation. Although it appears now to
be established that ribosomes lose the ability to reinitiate translation during
translation, a comprehensive investigation into the dependence on the various
initiation factors is still missing. For this, the truncated actin – luciferase series
of constructs can be used to isolate posttermination ribosomes at different
stages. Crosslinked at an intercistronic secondary structure, the associated
(initiation) factors could be analysed, e.g. by co-immunoprecipitation. Likewise,
by varying the length of the intercistronic spacer, reassociation of the factors
could be observed.
110
4. Discussion
In another aspect of this work, it was shown that uORFs can reduce
downstream initiation to an unexpected high extent when ribosomes are also
able to initiate and terminate within the uORF via 'leaky scanning'. These
ribosomes appear to block scanning or elongating ribosomes in a manner
similar to ribosomes stalled at a uORF termination site. It would be worthwhile
to investigate the occurrence of this phenomenon on mRNAs with 'wild type'
uORFs and whether it is used to regulate gene expression on these transcripts.
111
5. References
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