upstream elements present in the 3’utr of collagen genes … · the efficiency of 3’ end...
TRANSCRIPT
Upstream elements present in the 3’UTR of collagen genes influence the processing
efficiency of overlapping polyadenylation signals
Barbara J. Natalizio, Luis C. Muñiz, George K. Arhin+, Jeffrey Wilusz+ and Carol S. Lutz*
Department of Biochemistry and Molecular Biology, + Department of Microbiology and
Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ 07103
Running title: Regulation of human collagen pre-mRNA polyadenylation
* Corresponding Author
MSB E671, 185 S. Orange Ave. UMDNJ-NJMS Newark, NJ 07103 973/972-0899 973/972-5594 FAX [email protected] Key words: polyadenylation, collagen, upstream element, pre-mRNA processing
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on August 27, 2002 as Manuscript M208070200 by guest on M
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Abstract
3’ untranslated regions (UTRs) of genes often contain key regulatory elements involved in
gene expression control. A high degree of evolutionary conservation in regions of the 3’UTR
suggests important, conserved elements. In particular, we are interested in those elements
involved in regulation of 3’ end formation. In addition to canonical sequence elements,
auxiliary sequences likely play an important role in determining the polyadenylation efficiency
of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the
AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate
that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation
of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of
competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We
have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3’ end
formation. Close inspection of the COL1A2 3’UTR reveals an unusual feature of two closely
spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs
are important auxiliary polyadenylation elements in mammalian genes.
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Introduction
Poly(A) tails are found on the 3’ end of nearly every fully processed eukaryotic mRNA.
The poly(A) tail has been suggested to influence mRNA stability, translation, and transport
(reviewed in 1-4). Polyadenylation is a two-step process that first involves specific
endonucleolytic cleavage at a site determined by binding of polyadenylation factors (reviewed
in 5-10). The second step involves polymerization of an adenosine tail to an average length of
approximately 200 residues. These steps are tightly coupled processes since reaction
intermediates are not detectable under normal conditions.
The vast majority of eukaryotic polyadenylation signals contain the consensus sequence
AAUAAA between 10 and 35 nucleotides upstream of the actual cleavage and polyadenylation
site. In addition, sequences 10-30 nucleotides downstream of the cleavage site are known to be
involved in directing polyadenylation (11-13 and references therein). These downstream
elements (DSEs) can be characterized as a block containing 4 out of 5 uracil (U) residues.
These two sequence elements recruit CPSF (cleavage and polyadenylation specificity factor)
and CstF (cleavage stimulatory factor) respectively in order to define the cleavage site;
therefore mutations within these sequences abolish polyadenylation.
The intricate nature of this process implies that polyadenylation might be a useful
mechanism to regulate gene expression. The efficiency of 3’ end processing is a level at which
regulation can occur. Since most pre-mRNAs in the cell are not efficiently processed, even
small changes in the overall processing efficiency of a particular pre-mRNA may have a
substantial effect on gene expression. Experimental evidence has demonstrated that poly(A)
signal strength directly influences the amount of mature, exported mRNA (14, 15). Poly(A)
signal strength is also directly correlated with the rapid assembly of the polyadenylation
machinery on the nascent transcript (16) and with transcription termination efficiency (17).
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Detailed mechanistic studies on regulation of polyadenylation are now emerging, revealing
both cis- and trans-acting factors (reviewed in 5, 9, 18).
In addition to the sequence elements previously described, elements upstream of the
AAUAAA sequence (see below), and downstream of the DSE (see 19, 20 and references
therein), have been identified as auxiliary cis-acting polyadenylation efficiency elements. Such
elements may play an important role in modulating the overall processing efficiency. Upstream
elements (USEs) have been characterized in viral and cellular systems, including SV40 (21),
HIV (22-27), adenovirus major late region (28, 29), cauliflower mosaic virus (30), ground
squirrel hepatitis virus (31, 32), and human C2 complement (33, 34). Spacing between the
AAUAAA and the USEs plays a significant role in that the USEs closest to the AAUAAA are
most important (21). Studies on HIV suggest that definition of the polyadenylation site involves
the recognition of multiple sequence elements, including the USE, in the context of the
AAUAAA (27).
Comparisons between the polyadenylation signals of the SV40 late mRNA and other
cellular mRNAs revealed that three human collagen genes, COL1A1, COL1A2, and COL2A1,
each possess elements similar in sequence (USE consensus UAU2-5GUNA) and position
relative to the AAUAAA to the SV40 late USEs (21; C.S.L., unpublished data). Collagens are
extracellular proteins that are responsible for the strength and flexibility of connective tissue.
They account for 25-30% of all proteins present in animals, and are the major fibrous element
of skin, bone, tendon, cartilage, blood vessels, and teeth (see 35 and references therein). In
addition to their structural role, collagens have a directive role in tissue development. The basic
structural unit of collagen consists of three polypeptide chains that are extensively covalently
cross-linked to each other. The composition of the chains depends on the type of collagen. Type
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I collagen consists of 2 COL1A1 chains and 1 COL1A2 chain, whereas type II collagen
consists of 3 COL2A1 chains.
Interestingly, COL1A1, COL1A2, and COL2A1 each possess 3’ UTRs that are
extremely conserved between human and other vertebrates, including mice, cows, chickens and
pufferfish (36-43, see also GenBank and Table 1). For example, human COL1A1 has a 3’UTR
~1.5 kb in length with 2 polyadenylation signals. The first ~ 500 bases, containing the first
polyadenylation signal, are 86.5% identical in mouse, followed by a ~600 base block of little
conservation, and the final ~400 bases, including the second polyadenylation signal, are 71.1%
identical (44). This high degree of evolutionary conservation suggests important regulatory
functions of the collagen 3’UTRs. It is important to note that the use of one polyadenylation
signal over another will shorten the 3UTR, and this shortening could potentially remove
important regulatory elements. When the sequences surrounding the polyadenylation signal are
carefully examined, the percent identity is even higher, especially in the case of the final
polyadenylation signal (see Table 1). Mechanisms for alternative polyadenylation have been
extensively studied for the calcitonin and IgM genes (45-47), however, it is not mechanistically
understood how one collagen polyadenylation signal is chosen from among several.
Upregulation of collagen gene expression takes place in a variety of diseases,
including osteoarthritis and scleroderma, but it is unclear how this regulation of expression is
accomplished (48-51). Some studies have suggested in scleroderma that collagen production
may be upregulated by increased mRNA transcription (47, 52) but the altered expression may
not be fully explainable by changes in transcription rates and may additionally be
accomplished by regulated post-transcriptional mechanisms, such as polyadenylation.
This study examines the regulation of 3’ end formation in human collagen genes. The
strong evolutionary conservation of the 3’ UTRs, particularly around polyadenylation signals,
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led us to believe that these regions contained key regulatory elements. We asked whether cis-
acting USEs function as auxiliary elements to influence polyadenylation of the collagen
mRNAs, whether these elements acted in a similar fashion to other defined USEs, and how
these elements affect utilization of overlapping polyadenylation signals. We determined that
the USEs present in these collagen genes do influence 3’ end formation efficiency in these
genes. Furthermore, the organization of alternative poly(A) signals in the COL1A2 gene
suggests that assembly of 3’ end processing factors on the distal signal prohibits assembly of
the processing complex on the proximal signal. This suggests that protein-RNA interactions
between core polyadenylation signal elements may represent a novel method of downregulating
polyadenylation signal usage.
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Materials and Methods
In vitro transcription of RNA substrates
RNA transcripts for in vitro polyadenylation and cleavage reactions were synthesized
by use of SP6 RNA polymerase according to the supplier (Promega) in the presence of 50µCi
of [32P] UTP (Amersham Pharmacia Biosciences or Perkin Elmer Biosciences). Transcription
of COL1A2 yielded a 311 base RNA, of COL2A1 a 323 base RNA, and of COL1A1 a 274
base RNA. RNAs were gel-purified from 5% polyacrylamide-8M urea gels by overnight crush
elution in high salt buffer (0.4 M NaCl, 50 mM Tris at pH 8.0, 0.1% SDS) prior to use in
reactions. Eluted RNAs were ethanol precipitated and resuspended in water.
Nuclear extracts and in vitro polyadenylation and cleavage reactions
HeLa cell nuclear extracts were prepared as described previously (53). In vitro
polyadenylation reactions contained a final concentration of 58% v/v HeLa nuclear extract,
16mM phosphocreatine (Sigma), 0.8mM ATP (Amersham Pharmacia), 2.6% polyvinylalcohol,
and 1 x 105 cpm of 32P-labeled substrate RNA (approximately 50 fmol) in a reaction volume of
12.5µl (54, 55). Reactions were allowed to incubate at 30°C for 1 hour. Reaction products were
then extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and analyzed
on (19:1) 5% polyacrylamide gels containing 8M urea. Typical in vitro cleavage reactions
contained a final concentration of 58% v/v HeLa nuclear extract, 1mM cordycepin (Sigma),
0.5mM ATP, 20mM phosphocreatine, 2.6% polyvinylalcohol, and 1 x 105 cpm of [32P]-labeled
substrate RNA in a reaction volume of 12.5µl (56). Cleavage reactions were allowed to
incubate at 30°C for 1 hour. Products were then processed and analyzed as described for
polyadenylation products. Competition reactions were performed by adding increased
concentrations of specific or nonspecific oligoribonucleotides as indicated into the typical
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polyadenylation reaction mixtures and were allowed to proceed as described above. Reactions
were quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software.
Oligonucleotides
Oligonucleotides were synthesized on the Applied Biosystems 392 and 394 DNA/RNA
synthesizers in the New Jersey Medical School Molecular Resource Facility (Newark, NJ). A list of
the primers used in PCR reactions and cloning is found in Table 1. An oligoribonucleotide
representing the putative USE motifs of COL1A2 had the sequence
AUUAAAUUGUACCUAUUUUG. A nonspecific oligoribonucleotide was also synthesized and had
the sequence GUCACGUGUCACC.
Transfection and RNase protection
Human 293T and HeLa cells were maintained in Dulbecco’s Modified Eagles Medium
(DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS; Sigma) and
1% penicillin-streptomycin (P/S; Life Technologies). Cells were seeded in 100mm plates
approximately 12hrs prior to transfection. When cells reached 80% confluency, they were
transfected using the Quantum Prep Cytofectene reagent (Bio-Rad). Plasmid DNA (8.4 ug)
was diluted in 700ul of serum-free medium to which 40 ul of Cytofectene was added and the
mixture was incubated at room temperature for 20min. Following the addition of 6.3ml of
medium (plus FBS) to the mixture, the medium on the cells was removed and replaced with
the entire transfection cocktail. The Cytofectene-DNA mixture was removed 6 hrs after
transfection and replaced with fresh medium. After 24 hrs, cells were washed with once with
phosphate-buffered saline (PBS). Cells were scraped and collected into 1ml PBS and
transferred into microfuge tubes. Cells were then centrifuged at 1000 RPM for 5min. The
PBS was aspirated and pellets were stored at -80oC for no more than 2 days.
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Total RNA was extracted from the cell pellet using the RNeasy Mini Kit (Qiagen)
according to the manufacturer’s spin protocol for isolation of total RNA from animal cells.
Probe RNA was prepared as described above, using T7 polymerase and generating the
antisense of the CßS-COL1A2 construct. The reporter RNA levels were determined by RNase
protection using the RPAIII kit (Ambion Inc., Austin, Tex.) for 1 hr at 37?C. DNA templates
were then removed by DNase I digestion and the RNA was phenol extracted, ethanol
precipitated, and analyzed on 5% polyacrylamide 8M urea gels as described above.
Plasmids
COL1A2, COL2A1, COL1A1 inserts were generated by PCR using complementary
primers (Table 1). The COL2A1 and COL1A1 primers contained Bam HI (forward) and
Hind III (reverse) recognition sites to allow insertion into appropriately digested vectors;
whereas, the COL1A2 primers contained Bam HI (forward) and Pst I (reverse) recognition
sites. Gel purified and appropriately digested PCR fragme nts were ligated into appropriately
digested pGEM4 with T4 DNA ligase (Invitrogen) at 17°C overnight. The constructs were
then transformed into E. coli XL1-Blue cells. Positive clones were both sequenced and
assayed for expression of appropriately sized clones. Sequencing was completed by the New
Jersey Medical School Molecular Resource Facility using the Applied Biosystems 373 DNA
Sequencer, and resulting sequences were analyzed by BLAST computer programs for
accuracy. Amplification reactions were performed using Platinum Taq Polymerase
(Invitrogen) in a total volume of 50µl using standard reaction mixtures for 35 cycles of 95°C
(1 min), 55°C (30 sec), and 72°C (45 sec) with an initial denaturation step of 95°C (5 min).
PCR products were purified on a 1% agarose gel prior to use. Primers used in individual PCR
reactions are referred to in Table 1.
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The COL1A2 double mutant 1,2 and the triple mutant 1,2,3 were prepared via the
Stratagene Quick-Change Kit. Amplification reactions using Pfu Polymerase were for 20
cycles of 95°C (30 sec), 55°C (1 min), and 68°C (8 min) with a 95°C (5 min) initial
denaturation step. To remove the remaining template following amplification, a Dpn I
digestion was performed for 90 minutes at 37°C. The PCR product was then transformed into
E. coli XL-1 Blue cells. Positive clones were isolated and sequenced to determine correct
expression before use in polyadenylation reactions.
The PA2-G mutant COL1A2, the single USE mutants, and the double mutant 1,3 were
created by the use of the me ga-primer method of PCR mutagenesis as described previously
(57). Briefly, a mutant PCR product was generated using an internal upstream primer
containing the mutant, such as the PA2-G mutant COL1A2, and the wild-type COL1A2
downstream primer. This product containing the mutant sequence was then gel purified and
used as the downstream mega-primer for the second PCR. The COL1A2 wild type upstream
primer was used for the second PCR. A third PCR was then performed using the gel purified
second PCR product as the template and COL1A2 wild type upstream and downstream
primers. This final PCR amplified the inefficient product resulting from the second PCR. This
final product was gel-purified, cloned into pGEM4, and transformed into E. coli XL-1 Blue
cells.
The construction of pIVA2 was described in Wilusz et al. (58). Briefly, the 155 base Bam
HI to Pvu II fragment of pAd5-E1B (which contains adenovirus type 5 sequences from 3943-
4122) was cloned into pGem4 at the Hinc II and Bam HI sites. Linearization with Bgl I
yields a 158 base RNA containing the IVA2 poly(A) signal. The DNA template to generate
IVA2-USE RNA, which contains a USE 5' of the AAUAAA, was constructed by a two step
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PCR reaction using the megaprimer approach (57). The first PCR reaction used a standard
SP6 primer and
5'-TATTTAGGGGTTTTGCGGGTTACAAATAAAGCCGCGCGGTAGGCCGG to generate
a megaprimer that contained a USE insertion 13 bases upstream of the AAUAAA element.
The second PCR reaction contained the megaprimer and the primer
5'-AGCTTGCATGCCTGCAGGTCGACTC. The product of this PCR reaction was then cut
with Bgl II and used as a template to generate IVA2-USE RNA using SP6 RNA polymerase.
For transfection assays, all COL1A2 constructs were cloned into the Bam HI-Pst I
sites of vector pCßS (gift of David Fritz, UMDNJ) downstream of a CMV promoter and
upstream of a bovine growth hormone polyadenylation signal. This vector also includes intron
1 of the rabbit ß-globin gene accompanied by the splice donor and acceptor sites. Constructs
were verified by sequencing.
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Results
USEs can stimulate in vitro 3’ end processing of a weak polyadenylation signal.
Previously, auxiliary polyadenylation elements known as upstream efficiency elements
(USEs) have been described and characterized in the SV40 late polyadenylation signal (21). It
was determined that these elements functioned as efficiency elements in the SV40 system since
their disruption resulted in reduced polyadenylation function (21). It has also been shown that a
USE from the SV40 signal can replace the HIV USE in mediating efficient 3’ end formation in
transient transfection assays (23). Both SV40 and HIV have very strong polyadenylation
signals. However, it has not previously been determined if USEs could be added to a weak
polyadenylation signal, such as the adenovirus IVA2 polyadenylation signal, to enhance 3’
processing efficiency. A USE motif from SV40 having the sequence
GCUUUAUUUGUAACC was inserted upstream of the AAUAAA in the IVA2
polyadenylation signal to create IVA2-USE, substrate RNAs were prepared from both pIVA2
and pIVA2-USE, and the RNAs were added to in vitro polyadenylation reactions. Figure 1
shows that the presence of a USE in IVA2-USE enhanced polyadenylation efficiency
approximately four-fold as compared to IVA2 alone. These data indicate insertion of a USE can
increase polyadenylation efficiency of a weak processing signal, suggest that USEs can
modulate poly(A) site definition, and suggest that USEs may be commonly found in cellular,
not only viral, genes. It is important, therefore, to evaluate mammalian polyadenylation signals
for USE elements.
USEs can be found in collagen genes.
A survey of numerous cellular polyadenylation signals revealed that elements
resembling the USE motifs present in SV40 can also be found in many 3’UTRs; that is, similar
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to the consensus UAU2-5GUNA and within 75 bases of the AAUAAA (CSL, unpublished
data). We chose to focus on three collagen genes since their 3’ UTRs are highly conserved
through evolution, suggesting regulatory function. Figure 2A shows the comparison of the
SV40 late polyadenylation signal to three human collagen genes for type I (COL1A1 and
COL1A2) and type II (COL2A1) collagens. The polyadenylation signals AAUAAA/
AUUAAA and the putative USE elements are highlighted. We next wanted to determine if
these putative USEs present in the collagen 3’ UTRs could stimulate 3’ end processing
efficiency like the SV40 USEs. Previously it was shown that polyadenylation reactions
containing an SV40 substrate RNA could be inhibited specifically by oligoribonucleotides
representing the USE motifs (54). Plasmids encoding a portion of each collagen gene 3’UTR
containing the polyadenylation signals were created by PCR of human genomic DNA.
Substrate RNAs for the polyadenylation reactions were prepared by in vitro transcription using
SP6 polymerase in the presence of [32P] UTP. In vitro coupled cleavage and polyadenylation
reactions were performed using HeLa nuclear extract and COL1A2 (Figure 2B) substrate
RNA. Oligoribonucleotides representing a putative USE corresponding to COL1A2 or a non-
specific oligoribonucleotide were also added to the reactions. The results for COL1A2 are
shown as an autoradiogram of a typical in vitro polyadenylation reaction. The second lane in
Figure 2B, marked “0”, represents a reaction performed in the absence of competitor
oligoribonucleotides and demonstrates that the COL1A2 substrate RNA was efficiently
polyadenylated in our in vitro system. Similar results were found with COL1A1 and COL2A1
substrate RNAs and their specific oligoribonucleotides (data not shown). In each case, the
specific USE oligoribonucleotide inhibited polyadenylation, while the non-specific had no
effect on polyadenylation (see Figure 2B and data not shown). No effect was also noted when a
different non-specific oligoribonucleotide was used for each substrate RNA (data not shown).
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Quantitation of the percent polyadenylated product is indicated below the autoradiogram of the
gel in Figure 2B. Additionally, oligoribonucleotides representing the collagen USEs can cross-
compete in this assay (i.e. a COL1A1 oligo can compete with a COL1A2 substrate RNA; data
not shown). Taken together, these data suggest that the oligoribonucleotides specifically bind
and sequester common factor(s) important for polyadenylation, and suggest that the similarity
to the SV40 motifs identified in the collagen 3’ UTR is functionally significant.
COL1A2 USEs act as auxiliary polyadenylation elements in vitro.
Because of the strong processing efficiency observed using the COL1A2 substrate RNA, we
chose to focus our attention on that polyadenylation signal (diagrammed in Figure 3A). We
were also intrigued by the high degree of sequence conservation of this signal from diverse
organisms (see Figure 3A). We next made a series of substitutions replacing the COL1A2
USEs with Bgl II linkers in order to assess the contribution of the USEs to in vitro
polyadenylation (diagrammed in Figure 3B). USEs were replaced individually, as well as two
at a time and three at a time. A non-specific mutation was created by introducing a Bgl II linker
in a non-USE containing region upstream of the polyadenylation signal. RNAs were prepared
from each construct by in vitro transcription in the presence of [32P] UTP, were gel purified,
and were added to in vitro polyadenylation reactions using HeLa nuclear extract. Reaction
products were analyzed on 5% polyacrylamide 8M urea gels. The data were quantitated from
multiple in vitro reactions and are presented in Figure 3. Mutation of either USE 1, 2 or 3 alone
had little effect on polyadenylation. Mutation of both USEs 1 and 3 simultaneously also had
only a slight effect but co-mutation of USEs 1 and 2 or USEs 1, 2, and 3 diminished
polyadenylation efficiency to approximately half of wild type levels. A non-specific mutation
outside the USE region (NS mut) had no effect on polyadenylation. We conclude that none of
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the USEs are absolutely required for COL1A2 polyadenylation, but that mutation of USE 2 in
conjunction with at least one other USE led to the most dramatic decreases in polyadenylation.
COL1A2 USE mutations are more deleterious in in vivo assays.
Since all of our experiments so far have been performed in vitro, we found it important to
verify our results in in vivo assays. We next cloned our COL1A2 wild type and mutant
constructs into pCßS downstream of a CMV promotor and upstream of a BGH poly(A) signal.
Tandem polyadenylation signals have been used previously to examine requirements for a
different type of auxiliary sequences in the lamin B2 gene (59). A T7 promoter on the opposite
strand downstream of the BGH poly(A) signal was also present for ease in making antisense
probes. The constructs were then transfected into HeLa or 293T cells, and after 24 hours, total
RNA was harvested. This RNA was added to RNase protection assays (using an antisense
transcript from the T7 promoter as a probe). Representative RNase protection assays are shown
in Figure 4A, and the results of all our experiments were quantitated and are shown in Figure
4B. The results show that use of the COL1A2 polyadenylation signal prevailed over use of the
BGH polyadenylation site when the COL1A2 signal was wild type (lane 2) or had a non-
specific mutation (lane 7) but the USE mutations altered this ratio (lanes 4-6, 8-10). The
quantitated results were analyzed as the ratio of the protected RNA fragment corresponding to
RNA polyadenylated at the COL1A2 site relative to those polyadenylated at the BGH poly(A)
site (Figure 4B). A large number means that the COL1A2 polyadenylation signals were
preferentially used rather than the BGH polyadenylation signal, while a small number means
that the BGH polyadenylation signal was preferentially used. The overall trends in the in vivo
data correlate with the in vitro data; however, the USE mutants are more deleterious in varying
degrees in vivo as compared to in vitro. USE 1 and 3 mutations alone had little effect on use of
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the COL1A2 polyadenylation signal, whereas USE 2 mutation decreased polyadenylation
efficiency to approximately half of wild type. Mutation of the USEs in duplicate or triplicate
also reduced polyadenylation efficiency to approximately half of wild type.
COL1A2 has unusual, overlapping, competing polyadenylation signals.
Close examination of the COL1A2 mRNA sequence revealed an unusual feature, that
there are in fact two polyadenylation signals within 15 bases of each other (see Figure 3A).
Based upon the composition and spacing of the downstream CstF binding site (also known as
the DSE) relative to the AAUAAA, it might seem that poly(A) signal 1 would be preferentially
used instead of poly(A) signal 2. In order to formally investigate the question of which poly(A)
signal was the major site of polyadenylation, we turned to cleavage assays using cordycepin, a
non-hydrolyzable analog of ATP. It turns out that poly(A) signal 2 is the major site of
polyadenylation, while poly(A) signal 1 is the minor site (Figure 5A). When a non-usable
mutant of poly(A) signal 2 was created (AAUAAA to AAGAAA; PA-2 G), polyadenylation
now switched to poly(A) signal 1 (5A). This suggested that perhaps something more than
USEs and sequence spacing of the AAUAAA relative to the DSE is influencing poly(A) signal
choice in this system.
We then wanted to know how mutation of the USEs in combination with the poly(A)
signal 2 mutant (PA-2 G) affected 3’ end processing. In our in vitro assays, mutation of the
poly(A) signal 2 consensus hexamer from AAUAAA to AAGAAA did not affect the overall
polyadenylation efficiency (see Figure 3B, PA-G mut). In our in vivo RNase protection assays,
mutation of poly(A) site 2 had no effect on overall polyadenylation, but that mutation in
conjunction with the double and triple USE mutations decreased polyadenylation to
approximately one-fifth of wild type (see Figure 4A, lanes 2-3, 11-12 and Figure 5B). Taken
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together, these data demonstrate that USEs influence 3’ end formation efficiency in the
COL1A2 gene.
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Discussion
In this study we have identified auxiliary 3’ end processing elements in highly conserved
regions of the 3’ UTRs of human collagen genes. These elements promote efficient
polyadenylation in vitro and in vivo. In addition, COL1A2 has the unusual feature of
overlapping polyadenylation signals, one of which predominates, and suggests a novel
mechanism for poly(A) signal downreuglation (see below). These findings provide initial
insight into regulation of collagen gene expression that will hopefully aid our understanding
of disease initiation.
Human type I and type II collagen genes all have highly evolutionarily conserved 3’UTRs.
Indeed, the functional importance of the collagen 3’ UTRs is implied by their conservation. The
3’ UTRs likely contains important regulatory sequences that influence polyadenylation site
utilization, and may also ultimately influence the cytoplasmic fate of the mRNA. Recognition of
two core cis-acting elements (the AAUAAA and the downstream U-rich element) by the
polyadenylation factors CPSF and CstF is the key determinant of mRNA processing efficiency.
In the case of large 3’ UTRs and/or multiple polyadenylation signals, additional auxiliary
elements may be necessary to ensure proper polyadenylation. The 3’UTRs of these three
collagen genes likely require such auxiliary motifs to support the efficient assembly of
polyadenylation factors. Interestingly, within the evolutionarily conserved regions of these 3’
UTRs exist elements containing close homology with the USE auxiliary polyadenylation
elements of SV40. We show here that these USEs in the collagen 3’UTRs act as auxiliary
polyadenylation efficiency elements, and that these USEs play an important role in an
overlapping polaydenylation signal.
Our in vivo data suggest that the USEs might be most important for poly(A) signal 1
utilization since mutation of the USEs affects polyadenylation at that site more than when both
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poly(A) signals are intact. Our in vitro data also support this, although the effects are not as
dramatic (data not shown). As has been appreciated more completely in recent years, 3’ end
formation is interconnected both to the other major RNA processing events, splicing and
capping, and also to mRNA transcription (reviewed in 6, 9, 60). This interconnection likely
results in most efficient utilization of cis- and trans-acting signals. Thus, it is reasonable to
expect that the in vivo data most closely mimic regulation at the cellular level, and reflect the
co-transcriptional nature of these processes.
The overlapping polyadenylation signals present in the COL1A2 3’UTR are unusual. Our
data demonstrate that poly(A) signal 2 is the major site of polyadenylation while poly(A) signal
1 is used, but to a lesser extent (see Figure 5A). These data suggest a model, shown in Figure 6.
The configuration of the overlapping signals sets up a competition between CstF binding to
poly(A) signal 1 versus CPSF binding to poly(A) signal 2. Mutation of the AAUAAA in
poly(A) signal 2 activates usage of poly(A) signal 1 (see Figures 5A and 6). These data suggest
that the two polyadenylation signals are in competition with each other. Steric hindrance ma y
not allow the interaction between CPSF and CstF bound at the corresponding sites for poly(A)
signals 1 and 2 simultaneously, or it may suggest that CPSF-RNA interactions are dominant
over CstF interactions at the DSE for poly(A) signal 1. These data demo nstrate a principle that
protein-RNA interactions can interfere with scaffold assembly, suggesting a novel mechanism
for repressing poly(A) signal usage. It remains to be seen whether this arrangement could be
used to decrease polyadenylation efficiency at selected signals.
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Acknowlegements
The authors wish to thank the members of the Lutz, Wilusz, and O’Connor laboratories for helpful
experimental suggestions and comments on the manuscript. This work was funded by American
Cancer Society grant RPG-00-265-01-GMC, an Arthritis Investigator Award 2AI-LUT-A-5, and
an Arthritis Foundation, New Jersey Chapter grant 3AI-LUT-A to C.S.L., and NIH grants
CA80062 and GM63832 to J.W.
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Figure Legends
Figure 1 Inclusion of a USE stimulates in vitro processing of a weak polyadenylation
signal. Top, schematic of constructs used, including relative positions of the AAUAAA and
upstream element (USE). An arrow marks the cleavage site. Bottom, in vitro polyadenylation
reactions. 0 minutes, unreacted substrate RNA; 30 minutes, reaction products. Polyadenylated
products are noted as poly(A)+ on the right. Quantitation of per cent polyadenylation is noted
at the bottom. Percent polyadenylation was calculated as the quantitation of the
polyadenylated product divided by the total quantitated RNA in the lane.
Figure 2 Competition studies suggest USE-binding factors influence the processing
efficiency of collagen polyadenylation signals.
2A Sequence of SV40 late polyadenylation signal compared to three human collagen genes,
COL1A1, COL1A2, and COL2A1. Canonical AAUAAA or AUUAAA elements are shown in
bold, putative auxiliary upstream elements are underlined and italicized.
2B In vitro polyadenylation reactions using COL1A2 as substrate RNA. COL1A2 specific or
non-specific competitor oligoribonucleotides (see Table 2) were added to the reaction in the
amounts indicated at the top of the lane. Percent polyadenylation was calculated as the
quantitation of the polyadenylated product divided by the total quantitated RNA in the lane.
Figure 3 Substitution of multiple COL1A2 USEs affects polyadenylation efficiency.
3A Schematic of distal (poly(A) signal 1) and proximal (poly(A) signal 2) polyadenylation
signals. USEs are shown in striped boxes; canonical AAUAAA and corresponding
downstream CstF binding sites are shown. Cleavage sites are marked with arrows. Regions of
evolutionary conservation are noted at the bottom.
3B Schematic of COL1A2 USE mutant constructs (left) and percent polyadenylation of each
as observed in in vitro processing reactions (right). Polyadenylation was normalized to 100%
as wild type. Black boxes indicate substitution with a Bgl II linker.
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Figure 4 USEs influence in vivo polyadenylation efficiency of the COL1A2 signal.
4A Representative RNase protection assay, using HeLa cells. Bands marked as ? represent
those fragments protected when the BGH polyadenylation signal was used, and therefore
represent polyadenylation at that signal; bands marked as * represent those fragments
protected when the COL1A2 polyadenylation signal was used and therefore represent
polyadenylation at that signal. Because of the mutations created, these fragments were often
different in size and are diagrammed for ease of interpretation on the right side of the figure.
Lanes: 1, marker, pBR322 cut with MspI and 5’ end labeled with ?[32P]-ATP; lanes 2-12,
COL1A2 mutant or wild type constructs cloned into pC?S as indicated above the lane; lane
13, pC?S vector alone; lane 14, probe used for RNase protection.
4B Lighter gray bars, 293T cell transfections; darker gray bars, HeLa cell transfections.
Percent polyadenylation was measured as the ratio of COL1A2 polyadenylation site
utilization to the downstream bovine growth hormone polyadenylation site utilization as
quantitated by RNase protection assays. Large numbers represent COL1A2 polyadenylation
preferentially; small numbers indicate BGH polyadenylation preferentially. Constructs are
indicated on the X axis; see also Figure 3B.
Figure 5 COL1A2 has the unusual feature of two closely spaced, competing
polyadenylation signals.
5A In vitro cleavage assay reveals poly(A) signal 2 is the predominantly used polyadenylation
signal, while poly(A) signal 1 is a minor signal. Mutation of poly(A) signal 2 from AAUAAA
to AAGAAA switches this predominance. Marker lane, transcript prepared from COL1A2
construct that was linearized with Nsp I (cuts between the two cleavage sites).
5B In vivo polyadenylation assays reveal that USE mutants plus mutation of poly(A) signal 2
results in a marked decrease in polyadenylation efficiency. Lighter gray bars, 293T cell
transfections; darker gray bars, HeLa cell transfections. Percent polyadenylation was
measured as the ratio of COL1A2 polyadenylation site utilization to the downstream bovine
growth hormone polyadenylation site utilization as quantitated by RNase protection assays.
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Figure 6 CPSF binding between the core elements of the proximal polyadenylation signal
of COL1A2 may inhibit complex assembly on the distal polyadenylation signal.
Polyadenylation machinery can successfully assemble on the proximal signal (poly(A) signal
2) but may not support assembly of processing factors on the distal signal (poly(A) signal 1)
due to spacing or steric constraints.
Table 1 Evolutionary conservation of collagen genes
Human COL1A1, COL1A2, and COL2A1 were examined for 3’ UTR size, number of
polyadenylation signals, and searched for evolutionary conservation of the 250-300 bases
surrounding the final polyadenylation signal of the cognate gene by BLAST. Accession numbers are
as follows: COL1A1: M55998, BTA312112, AY083537.1, AL606480.11; COL1A2: NM_000089.2,
AC091773, GGCOLA2C; COL2A1: XM_056481, AF023169, BTCOLII, RNAJ4879. COL1A2 is
listed as having 5 or 6 polyadenylation signals because one signal has the non-consensus sequence
AUUAA (42).
Table 2 Primers used in construct preparation by guest on May 15, 2020
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TABLE 1
Gene size of 3’ UTR # polyadenylation signals % sequence identity to final PA signal
COL1A1 ~1.5 kb 2 92% cow, 98% macaque, 92% mouse
COL1A2 ~900 bases 5 or 6 79% pufferfish, 84% chicken
COL2A1 ~400 bases 2 95% dog, 86% cow, 89% rat
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TABLE 2
Primers for Cloning
COL1A2:
forward primer: TAGGATCCAAGTATGCAGATTATTTG
reverse primer: TATACTGCAGGGCTGGTAGAGATGC
COL1A1:
forward primer: TAGGATCCGGGTTTCAGAGACAACTTC
reverse primer: TATAAAGCTTGCCCATCACCCCAAG
COL2A1:
forward primer: TAGGATCCGTCAAGGCAGAGGCAGGAAAC
reverse primer: TATAAAGCTTTCCTTAGGACTGCTATTTG
G mutants and truncated transcripts:
Hexamer Mutant COL1A2: TAAATTGTGAAAAAAATGAAAGAAAGCATGTTTGGT
COL1A2-370: TAGGATCCCAAAGTTGTCCTCTTCTTCAG
COL1A2-400: TAGGATCCATTTGTTCTTTGCCAGTCTC
COL1A2-455: TAGGATCCGTTTCTTGGGCAAGCAG
COL1A2-500: TAGGATCCATGTGAGATGTTTAAATAAATTG
Single USE Mutant Primers:
COL1A2 Mutant A: TCAGCATTTGTTCTTTGCCAGATCTCATTTTCATCT
COL1A2 Mutant 1: TTGGGCAAGCAGAAAAACTAAAGATCTCCTATTTTGTATAT
COL1A2 Mutant 2: GCAGAAAAACTAAATTGTACCTAGATCTATATGTGAGATG
COL1A2 Mutant 3: ATATGTGAGATGTTTAAATAAAGATCTAAAAAAATGAAATAAAGCAT
Double USE Mutant Primers:
COL1A2 Double Mutant 1,2:
TTGGGCAAGCAGAAAAACTAAAGATCTCCTAGATCTATATGTGAGATGTTTAAAT
COL1A2 Double Mutant Complement 1,2:
ATTTAAACATCTCACATATAGATCTAGGAGATCTTTAGTTTTTCTGCTTGCCCAA
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Barbara J. Natalizio, Luis C. Muniz, George K. Arhin, Jeffrey Wilusz and Carol S. Lutzefficiency of overlapping polyadenylation signals
Upstream elements present in the 3'UTR of collagen genes influence the processing
published online August 27, 2002J. Biol. Chem.
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