Ribosomal proteins L5 and L15 Functional characterisation of important features, in vivo

Ivailo Simoff

© Simoff, Ivailo, Stockholm 2009 ISBN 978-91-7155-896-1 Printed in Sweden by US-AB, Stockholm 2009 Distributor: Department of Cell Biology, Stockholm University

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The tighter you squeeze, the less you have. Zen Saying

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Abstract

Protein synthesis is a complex, highly regulated and energy consuming proc-ess, during which a large ribonucleoprotein particle called the ribosome, synthesizes new proteins, according to the specification laid down in the genes. The eukaryotic ribosome consists of two unequal parts called: small and large ribosomal subunits. Both ribosome subunits are composed of ribo-somal RNA (rRNA) and ribosomal proteins (r-proteins).

Although rRNAs build the matrix of the ribosome and carry out its main enzymatic function, catalysing the peptide-bond formation between amino acids, r-proteins also appear to play important structural and functional roles. The important functions of r-proteins in eukaryotes are underscored by the fact that most of the r-proteins are essential components of the eukaryotic ribosome. The primary role of r-proteins is to initiate the correct tertiary fold of rRNA and to organize the overall structure of the ribosome.

In this thesis, I focus on two proteins from the large subunit of the eu-karyotic ribosome: r-proteins L5 and L15 from bakers yeast Saccaromyces cerevisiae. Both r-proteins are essential for ribosome function. Their life cycle is primarily associated with rRNA interactions. As a consequence, the proteins show high sequence homology across the species borders. Further-more, both L5 and L15 are connected to various human diseases which makes it important to elucidate their role in ribosome biogenesis and ribo-some function.

By applying random- and site-directed mutagenesis, coupled with func-tional complementation tests, I aimed to elucidate functionally vital regions in both proteins, implicated in transport to the cell nucleus, protein-protein interactions and/or rRNA binding. The importance of individual and multi-ple amino acid exchanges in the primary sequence of rpL5 and rpL15 were studied in vivo. The results obtained in this study show that S. cerevisiae rpL15 was highly tolerant to amino acid exchanges in the primary sequence, whereas rpL5 was not. Consequently, A. thaliana ortholog to rpL15 could substitute for the function of wild type rpL15. In contrast, despite extensive similarities in amino acid sequence S. pombe’s ortholog could not substitute for the yeast S.cerevisiae counterpart. In comparison, different organisms’ rpL5 orthologs appear to be not interchangeable. Thus, orthologs in A. thaliana, D. melanogaster and M. musculus could not substitute yeast rpL5 in vivo. These observations further emphasize the importance of studying r-proteins as separate entities in the ribosome context.

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List of papers

This work is based on the following publications, which will be referred to by their Roman numerals

I. Ivailo Simoff, Hossein Moradi and Odd Nygård. Functional characterization of ribosomal protein L15 from Saccaromyces cerevisiae. Curr Genet. (2009) 55, 111-125.

II. Ivailo Simoff and Odd Nygård. Locating the nuclear localization signal in S. cerevisiae ribosomal protein L15A. (manuscript)

III. Hossein Moradi, Ivailo Simoff, Galyna Bartish and Odd Nygård.

Functional features of the C-terminal region of yeast ribosomal protein L5. Mol Genet Genomics. (2008) 280, 337-350.

IV. Ivailo Simoff, Hossein Moradi and Odd Nygård. Implications of N-terminal sequence elements in S. cerevisiae ribosomal protein L5. (manuscript)

Published papers are reproduced with permission from the publisher.

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Contents

1. Introduction .............................................................................................9

2. Protein synthesis ..................................................................................11 2.1. The ribosome .....................................................................................................11

2.1.1. Morphology ................................................................................................11 2.1.2. Function......................................................................................................12

2.2. The translation process....................................................................................13 2.2.1. Initiation .....................................................................................................13 2.2.2. Elongation ..................................................................................................14 2.2.3. Termination and recycling......................................................................15

3. Ribosomal components .......................................................................17 3.1. Ribosomal proteins ...........................................................................................17

3.1.2 r-protein genes (RPGs) ............................................................................18 3.1.3 r-proteins and pathology .........................................................................18 3.1.4. Nucleocytoplasmatic transport of r-proteins......................................19

3.2. Ribosomal RNA ..................................................................................................20 3.2.2 rRNA modifications....................................................................................21

3.3. Ribosome assembly ..........................................................................................22 3.3.1 r-proteins and ribosome assembly ........................................................23

3.4. Ribosome biogenesis and TOR regulation ...................................................23

4. YrpL15A ..................................................................................................26 4.1. General characteristics of YrpL15A ...............................................................26 4.2. YrpL15A in 60S complex..................................................................................26

4.2.1. Mutational analysis on YrpL15A Functional implications .................28 4.3. Paralogues of L15 genes in yeast and promoter functions......................29 4.4. YrpL15A and NLS...............................................................................................31

5. YrpL5 .......................................................................................................33 5.1. General characteristics of the protein ..........................................................33 5.2 YrpL5 on 60S .......................................................................................................33 5.3. L5•5S rRNA complex ........................................................................................34 5.4. YrpL5 function ....................................................................................................35 5.5. Nuclear transport of rpL5•5S rRNA ...............................................................38

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5.6. Dominant negative effects of non-functional YrpL5 alleles on cell growth ..........................................................................................................................39

6. Discussion ..............................................................................................40 6.1. The role of the C-terminal tag........................................................................40 6.3. Sensitivity to mutations and organism orthologs ......................................41

7. Acknowledgement ................................................................................42

8. References .............................................................................................44

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Abbreviations

ArpL15B A. thaliana r-protein L15B CP Central protuberance of the ribosome eEF Eukaryotic elongation factor eIF Eukaryotic initiation factor ES Expansion segment GAP GTP-ase activating protein HmrpL15e H. marismortui r-protein L15e HmrpL7Ae H. marismortui r-protein L7Ae HurpL5 H. sapiens r-protein L5 LSU Large subunit of the ribosome NLS Nuclear localization signal NPC Nuclear pore complex PrpL15A S. pombe r-protein L15A PTC Peptidyl transferase centre RF Release factor RPG r-protein coding gene rpS or L r-protein from SSU or LSU, respectively r-proteins Ribosomal proteins rRNA Ribosomal RNA RRF Ribosome recycling factor snoRNA Small nucleolar RNA SSU Small subunit of the ribosome TOR Target of rapamycine YeEF2 S. cerevisiae elongation factor 2 YrpL15A S. cerevisiae r-protein L15A YrpL5 S. cerevisiae r-protein L5 YrpL8 S. cerevisiae r-protein L8

1. Introduction

Proteins are key components in all living organisms. For example they are structural components, chief providers of catalytic functions and regulators of metabolic processes in all living cells. Every protein molecule mirrors the complex information coded for by the genome. The linear information resid-ing in a gene is translated into a sequence of amino acids that folds into a 3-dimensional protein structure. A large molecular machine called the ribo-some catalyses this translation process. Ribosomes are universal cellular complexes that demonstrate remarkable structural and functional similarities across all the three domains of life.

The actual process of translation has three basic steps: initiation, elonga-tion and termination. At the end of the translation cycle the ribosome has build a polypeptide chain by assembling amino acids in the order specified by the translated mRNA. After completing the translation the ribosome is ready for a new round of protein synthesis.

Mature eukaryotic ribosome contains two unequal subunits: the small (SSU) and the large (LSU) subunit. The two subunits are built from ribo-somal proteins (r-proteins) and four different types of ribosomal RNA (rRNA). In yeast the SSU contains 18S rRNA and 32 r-proteins, whereas the LSU consists of 25S, 5,8S and 5S rRNA and 46 r-proteins. Approximately 40% of the ribosomes mass comes from r-proteins the rest is rRNA.

The ribosome assembly is a multi-step process that involves several com-partments in the eukaryotic cell. The initial phase in ribosome assembly starts in a special sub-compartment of the cell nucleus, called nucleolus. Here, the rDNA genes are transcribed to rRNA precursors. During transcrip-tion a plethora of modifying enzymes, protein factors and r-proteins are en-gaged in modification, maturation and folding of the rRNA transcript to build the pre-ribosome subunits. The nucleolus has a vectorial organization. Within it, assembly factors are situated in different compartments and aid the process of ribosome assembly, as the pre-ribosomes emerge. In all types of ribosomes, the rRNA carries out the main catalytic function of peptide bond formation, while r-proteins contribute to the structural stability of rRNA and aid the physiological processes completed by the ribosome. Despite several decades of investigation in the translation field, the role of r-proteins, with few exceptions, has remained in the shadow of rRNA function.

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In an attempt to bridge this gap in the current knowledge, the focus of this thesis pertains to the r-proteins, YrpL5 and YrpL15A, found in Saccharomy-ces cerevisiae LSU.

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2. Protein synthesis

2.1. The ribosome

2.1.1. Morphology Eukaryotic cells contain several types of ribosomes. Ribosomes are found in organelles such as mitochondria, chloroplast and in the cytoplasm. The latter category of ribosomes is hereafter referred to as eukaryotic ribosomes or 80S ribosomes.

Our current knowledge on the detailed structure of the ribosomes is mainly derived from high-resolution crystallographic studies on prokaryotic ribosomes (Ban et al. 2000; Schluenzen et al. 2000; Brodersen et al. 2002). In contrast the knowledge on the structure of eukaryotic ribosomes is mainly derived from low-resolution 3D-structures obtained by cryo-electron micros-copy (Gomez-Lorenzo et al. 2000; Spahn et al. 2001; Taylor et al. 2007). The eukaryotic and prokaryotic ribosomes share same morphological fea-tures. It is thought that the morphological resemblance between prokaryotic and eukaryotic ribosomes also implicates functional similarities (Spahn et al. 2001). The morphological relationship between eukaryotic and prokaryotic ribosomes stems from the closely identical secondary and tertiary structures of the folded rRNA components (Spahn et al. 2001). Irrespective of their origin, the ribosomes are composed of two subunits: the small ribosomal subunit (SSU) and the large ribosomal subunit (LSU). The SSU is divided in two parts, the head and the body, separated by the neck region (Verschoor et al. 1998), (Fig. 1). The body has a prominent protrusion called the platform. The LSU has three distinctive protrusions: the central protuberance (CP), the L1 and L7/L12 stalk (Fig. 1).

Despite the morphological similarities between ribosomes in prokaryotes and eukaryotes, the size of the ribosomes differs considerably. Eukaryotic ribosomes are 30% larger than their bacterial counterparts (Wilson et al. 2003). This size difference is partly due to an increased size of the main types of rRNA (16S-like and 23S-like rRNAs) and partly due to an increased number of r-proteins (Spahn et al. 2001). The additional nucleotides found in the rRNAs are inserted at specific positions in the rRNA core structures.

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Figure 1. Ribosome morphology representing the SSU (to the left) and LSU (to the right). Complete ribosome particle is in the middle. L1 protuberance, CP central protuberance, L7/L12 stalk. Figures A thru F represent ribosomal subunits in differ-ent orientations.

These additional sequence elements are referred to as expansion segments (ES) (Spahn et al. 2001; Wilson et al. 2003). Eukaryotic ribosomes contain some 20 more r-proteins compared to the bacterial counterparts (Smith et al. 2008).

In this thesis I will mainly deal with ribosomes from bakers yeast S. cere-visiae.

2.1.2. Function In the mature 80S ribosome, both subunits contribute to the translation

process. Interaction between the SSU and LSU results in formation of an intersubunit cavity in which the actual translation process takes place. This cavity holds the mRNA codons currently under decoding and the interac-tions sites for tRNA. The tRNA interaction sites are referred to as the ami-noacyl (A-), the peptidyl (P-) and the exit (E-) sites. The P- and A- sites can hold peptidyl-tRNA and/or aminoacyl-tRNA whereas the E- site accommo-dates deacylated tRNA. The correct physical proximity of the ribosome sub-

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units is ascertained by a number of intersubunit contacts or intersubunit bridges consisting mainly of RNA-RNA and RNA-r-protein interactions. Some of these bridges are present in all types of ribosomes irrespective of their origin (Spahn et al. 2001), while some contact sites are present only within eukaryotic ribosomes (Spahn et al. 2001). The ribosome bound ligands; such as tRNA also contribute to the intersubunit interaction.

The subunits play distinctive roles in the translation process. The primary role of SSU is in decoding the message in mRNA by assuring the correct base pairing (decoding) between the codon on mRNA and anticodon loop of tRNA (Schluenzen et al. 2000). The LSU catalyses peptide-bond formation between adjacent amino acids. A special site on LSU called peptidyl trans-ferase centre (PTC) is involved in building the peptide bond (Nissen et al. 2000). As this function is inherent to rRNA, with no apparent contribution of r-proteins in the catalytic process, the ribosome is a so-called ribozyme (Spahn et al. 2001), (Klein et al. 2004). The nascent polypeptide passes through a 100Å long channel that leads from the interface cavity to the back of the LSU. This channel is mainly composed of rRNA, with some portions of it containing r-proteins (Nissen et al. 2000).

The basal mechanism of translation is similar in all cell types. However, the compartmentalisation of the transcription and translation processes in eukaryotes increases the need for highly developed mechanisms for transla-tion regulation. This need is met by increasing the complexity in for example the initiation process in eukaryotes (Spahn et al. 2001). Another conse-quence of compartmentalisation is the need for transport of proteins e.g. r-proteins across the nuclear membrane (Spahn et al. 2001).

2.2. The translation process During the translation process, amino acids are incorporated into the

growing polypeptide at a rate of 10-20 amino acids per second (Wilson et al. 2003). The translation process is very accurate with one error occurring every 3000 amino acids incorporated (Wilson et al. 2003). In eukaryotes, the following steps are involved in the translation process: initiation, elongation, termination and recycling.

2.2.1. Initiation The initiation step starts with the disintegration of the 80S ribosome into its two constituent subunits, (for recent review see (Rodnina et al. 2009)). Bind-ing of eukaryotic initiation factors (eIFs) eIF 1, eIF1A and eIF3 to the SSU promotes the subunit separation. A specific initiator methionyl-tRNAi, in complex with eIF2 and GTP, is placed in the peptidyl (P-site) of the SSU, thus forming a 43S preinitiation complex. The mRNA to be translated is

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present in the cytoplasm as an mRNP particle, containing poly(A) binding protein (PABP) and eIF4F. This initiation factor specifically recognise the m7G cap and the poly(A)-tail of the mRNA. Interaction of the 43S pre-initiation complex with the mRNP particle leads to formation of new com-plex referred to as the 48S complex. This interaction is promoted by eIF3. As a result the 40S subunit is bound to the 5’-end of the mRNP-particle. The start codon AUG is reached by a scanning mechanism in which the 40S par-ticle, with its bound initiation factors and Met-tRNA, moves along the mRNA till it reaches the start codon. Upon AUG recognition eIF1and eIF2•GDP + Pi are released from the SSU under the simultaneous joining of the LSU to mRNA-bound SSU (Rodnina et al. 2009). The formed particle is referred to as a programmed 80S ribosome. In 80S ribosome, the Met-tRNA is positioned in the ribosomal P-site. Joining of the 40S and 60S subunits is promoted by eIF5B, in an energy dependent manner (for reviews see (Sonenberg et al. 2007; Rodnina et al. 2009; Sonenberg et al. 2009)).

2.2.2. Elongation The actual synthesis of the new protein takes place during the elongation step. At this stage an aminoacyl-tRNA, in complex with eukaryotic elonga-tion factor 1 (eEF1) and GTP, enters the A- site of the SSU. This occurs only if the universally conserved bases A 1492/1823 and A 1493/1824 in the 16S/18S rRNA (E. coli/S.cerevisiae numbering) can contact two of 2’ hy-droxyl groups in the mRNA codon (Yoshizawa et al. 1999). This interaction places the mRNA codon in correct orientation with respect to the anticodon on the cognate tRNA (Yoshizawa et al. 1999). When aminoacyl-tRNA is positioned in A-site of the LSU, a peptide bond is formed between the grow-ing peptide chain of the P-site bound peptidyl-tRNA (Met-tRNAi in the reac-tion following the initiation). The reaction, which involves proton donation from the α-NH2 group of the aminoacyl-tRNA to the carbonyl group of the peptidyl-tRNA (Nissen et al. 2000), results in a ribosome with peptidyl-tRNA in the A-site and a deacylated tRNA positioned in the P-site. The pep-tidyl-tRNA has to be moved back to the P-site to restore the ability of the A-site to accept the next aminoacyl-tRNA. The translocation, which is facili-tated by elongation factor 2 (eEF2), also involves the simultaneous move-ment of the deacylated tRNA from the P-site to the E-site and a movement of the ribosome relative to the mRNA by one codon, reviewed in (Fraser et al. 2007). This synchronized movement of the mRNA-tRNA complex occurs in the junction area between the subunits (Sengupta et al. 2008). There are different models for the elongation process. In the classical model (Kaziro 1978) the translocation of the ribosome precedes GTP hydrolysis. In con-trast, the updated model GTP hydrolysis is suggested to occur prior to ribo-some translocation (Rodnina et al. 1997).

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In yeast, an additional elongation factor eEF3 (Triana-Alonso et al. 1995) is involved in the release of the deacylated tRNA from the ribosomal E-site. Unlike the other nucleotide-binding translation factors eEF3 is an ATP bind-ing protein.

During the translation process several intermediate states of tRNA bind-ing to the ribosome have been reported (Frank 2001). The tRNAs move from the A-site and P-site bound state to E-site and P-site bound state, respec-tively, thus completing one round of the elongation cycle. The main factor in elongation step of translation is eEF2. eEF2 aids the elongation cycle by introducing conformational changes in the head-region of the SSU. These changes result in a ratchet-like movement of the head of SSU (Frank 2001) leading to translocation of tRNA-mRNA complex from P-, A- to E-, P-state. The tRNA-mRNA complex is finally released and the SSU ratchets back, thus completing the elongation cycle. The back ratcheting of the SSU is fa-cilitated by hydrolysis of GTP bound to eEF2, thus resulting in weakening of the established contacts during the forward ratcheting state (Sengupta et al. 2008). The forward and backward ratcheting are unequal processes, thus securing the one-codon movement of the mRNA. The ratcheting movement of the ribosome is presented in Fig. 2.

Figure 2. Ribosome translocation. (A-) aminoacyl, (P-) the peptidyl and (E-) the exit sites on the ribosome. (eEF2-GTP/GDP) eukaryotic elongation factor 2 in complex with GTP or GDP, respectively. Adopted from (Frank et al. 2007)

2.2.3. Termination and recycling The next step in the translation process is the termination step. Termination of the translation occurs when a stop codon is positioned in the A-site of ribosome. A special codon-dependent release factor (eRF1), positioned in the A-site, initiates hydrolysis of the ester bond, between the nascent peptide and tRNA, thus releasing the nascent polypeptide. An additional, codon-independent, GTP-binding factor eRF3 stimulates the release of the peptide in a GTP-dependent manner. The ability of eRF1 to bind to the A-site of the ribosome is enabled by its overall structure, which resembles that of tRNA (Inge-Vechtomov et al. 2003).

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The final step of the translation process is ribosome recycling. This step is quite different in bacteria compared to eukaryotes. In bacteria, a special ribo-some recycling factor (RRF) is present that promotes ribosome subunit dis-sociation. In eukaryotes, there is no ortholog to RRF; instead the current understanding of the process is that eIFs are responsible for subunit and mRNA dissociation (Rodnina et al. 2009).

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3. Ribosomal components

3.1. Ribosomal proteins During the 1970’s the widespread view was that r-proteins were the actual enzymatic components in the ribosome. The role of rRNA was to merely provide a structural scaffold for the r-proteins enzymatic activity. It was first in the beginning of the 1980’s Cech and colleagues discovered the catalytic activity of the RNA (Kruger et al. 1982). Since the discovery of catalytic RNA, ribosome research has largely been focused on rRNA and its role in ribosome functions (Kruger et al. 1982; Guerrier-Takada et al. 1983; Noller et al. 1995; Green et al. 1997; Steitz et al. 2003). Today the role of rRNA in the mRNA decoding process and in the peptidyltransferase reaction is widely acknowledged. Despite the pivotal role of rRNA in ribosome tasks, a growing interest for r-proteins and their contribution in ribosome function is now emerging (Green et al. 1997; Steitz et al. 2003; Dresios et al. 2006). In addition, the increased interest for studying r-proteins comes from growing evidences of r-protein implications in diseases e.g. cancer (Lindstrom 2009).

3.1.1 Function R-proteins are small, highly basic molecules. The high proportion of basic amino acids lysine and arginine enable them to counteract the negative charges of the phosphates in the backbone of the rRNA. Additionally, r-proteins are rich in glycine residues, which give them the needed flexibility for folding (Wilson et al. 2005). Many of the r-proteins have a globular do-main that is often exposed to the solvent side of the ribosomal subunits. The globular domain frequently contains acidic amino acids needed for interac-tion with additional components (Wilson et al. 2005). The primary role of eukaryotic r-proteins is to stabilize the interactions between different do-mains in rRNA (Ban et al. 2000; Spahn et al. 2001; Klein et al. 2004). Thus r-proteins provide the needed supporting scaffold for rRNA. In addition to the stabilizing role, r-proteins can have RNA chaperone activity (Wilson et al. 2005). The r-proteins can be divided into, early and late rRNA binders (Klein et al. 2004). Early r-proteins bind to the 5’ region of rRNA and have a large proportion of their surface area buried in the rRNA, thus providing for the initial entropy reduction in the emerging rRNA structure. Often these

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proteins have a globular part and one or several protrusions that penetrate deep into the rRNA. The morphology of the extended part is dependent on the interaction with rRNA (for a review see (Wilson et al. 2005)). In con-trast, late rRNA binding r-proteins do not burry such large surface area with rRNA, bind the 3’ end of rRNA and participate in extensive protein-protein surface interactions instead (Schluenzen et al. 2000; Klein et al. 2004).

3.1.2 r-protein genes (RPGs) Eukaryotic organisms often contain multiple copies of the genes coding for r-proteins. Plant cells contain three to five copies of each RPG. In animals, in general and humans in particular, single gene copy codes for every r-protein. In budding yeast, 59 of the 78 RPGs are present as doubles (Wolfe et al. 1997). The exact physiological meaning of these duplications is pres-ently not known. In contrast to rDNA genes that are located on one single chromosome, the RPGs are scattered throughout the S. cerevisiae genome.

Generally eukaryotic r-proteins are under regulation of strong promoters, generating excess amounts of r-proteins (Lam et al. 2007). Several transcrip-tion factors Fhl1, Ifh1 and Rap1 are associated with r-protein transcription regulation (Schawalder et al. 2004). Binding of these factors is regulated by several conserved signalling pathways (TOR, Ras/protein kinase A and C), (Schawalder et al. 2004)

3.1.3 r-proteins and pathology Apart from the structural contribution to ribosome function and stability, several r-proteins are involved in some diseases. In humans for instance, Diamond-Blackfan anaemia is correlated to mutations in rpS19, rpS24 and rpS17 (Draptchinskaia et al. 1999; Leger-Silvestre et al. 2005; Gregory et al. 2007). This syndrome is also linked to an increased risk for leukaemia (Wasser et al. 1978). Recent reports have shown the presumptive role of r-proteins for tumour formation in zebrafish and in mice (Beck-Engeser et al. 2001; Amsterdam et al. 2004). These tumours resulted from an inhibition of translation of p53 ((Warner et al. 2009) and references therein). Admittedly the most interesting finding is that ribosomal P-proteins were involved in neuropsychotic systemic lupus erythematosus (SLE) (Katzav et al. 2007). Apart from these pathological conditions several r-proteins have been linked to processes that apparently has little to do with the actual translation mechanism as endonuclease activity for P0 (Grabowski et al. 1991) and S3 in the DNA damage processing (Kim et al. 1995), L7 as co-activator of nu-clear receptors and L7, S20 and S3a in apoptosis (Goldstone et al. 1993; Neumann et al. 1997; Naora et al. 1998). In eukaryotic cells, e. g. S28, L2 autoregulate their own mRNA production (Warner et al. 2009). One of the most interesting observations implicating r-proteins L5 and L11 is the regu-

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lation of p53 level by their combined binding to murine ubiquitin ligase MDM2 (Warner et al. 2009). This can have implications in apoptosis and cancer.

3.1.4. Nucleocytoplasmatic transport of r-proteins The compartmentalization of the eukaryotic cell with transcription of and ribosome assembly occurring in the nucleus and the translation of mRNAs coding for r-proteins taking place in the cytoplasm necessitates substantial transport of molecules across the nuclear pore complex (NPC). The transport is facilitated by soluble receptors that are members of the karyopherin family proteins (type α or β) also known as importins (for review see (Mattaj et al. 1998) (Cook et al. 2007)). β-karyopherins can mediate nuclear import of a cargo molecule in two ways. The β-karyopherins can bind directly to a spe-cial amino acid sequence, termed nuclear localization sequence (NLS) that is part of the cargo molecule. The β-karyopherins can also interact with the cargo via an adaptor molecule of the importin-α type. The importin-complex formed is transported from the cytoplasm to the nuclear envelope, where the interaction occurs with the components of NPC known as nucleoporins. As-sociation of the RanGTP with the imported complex facilitates the cargo release inside the nucleus (Mattaj et al. 1998). RanGTP and RanGDP are unevenly distributed in the cell. In the cytoplasm RanGDP is in higher con-centration than in nucleus. The nucleotide state of Ran is regulated by addi-tional factors associated, RanGTP-ase activating protein (RanGAP),

As the ribosome assembly occurs in the nucleolar compartment of the nu-cleus, nuclear import of r-proteins from the cytoplasm is necessary to main-tain ribosome production. RanGTP-ase proteins and specific karyopherins facilitate the nuclear import of r-proteins (Moroianu 1998). R-proteins are directly transported to the nucleus without need for an adaptor molecule (Rout et al. 1997; Sydorsky et al. 2003) and Fig. 3. In S. cerevisiae the im-portin-β proteins Yrb4p/Kap123p, Pse1p/Kap121p, Sxm1p/kap108p and Nmd5p/Kap119p fulfil this function (Sydorsky et al. 2003). Apart from their transport function, r-protein specific β-karyopherins might also be important for preventing precipitation of the highly basic r-proteins (Jakel et al. 2002). How exactly the r-protein specific importin bind to the respective r-protein is presently not known. It is thought that the importin binds the cargo with its inner concave surface. The importin recognizes a special primary sequence of the cargo, the aforementioned NLS (Cook et al. 2007). The NLS is typi-cally composed of basic amino acids K and/or R. Dependent on the struc-tural features NLS can be divided in monopartite (Chelsky et al. 1989) and bi-partite NLS (Dingwall et al. 1991). In a classical monopartite signal a cluster of three basic amino acids is present within consensus sequence (K/R)3X1-4 (Timmers et al. 1999) and is often flanked by helix-braking G or P. A bi-partite signal contains two clusters of basic amino acids. One cluster

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Figure 3. Schematic representation of nuclear transport of r-proteins. (β) β-karyopherin, (RP) r-protein, (RanGTP) Ran GTP-ase protein, (NPC) nuclear pore complex.

conforms to the definition of a monopartite NLS and the other is a basic di-peptide (Dingwall et al. 1991; Stuger et al. 2000). The di-peptide cluster is situated upstream of the of the monopartite cluster. An intervening spacer divides both clusters. The length of the spacer varies but should be at least 10 amino acids long (Cook et al. 2007). R-proteins often contain more than one NLS (Kundu-Michalik et al. 2008).

3.2. Ribosomal RNA The ribosome is a ribonucleoprotein particle composed of four different types of rRNA. In budding yeast S. cerevisiae the four rRNAs are referred to as 18S, 25S, 5,8S and 5S rRNA. The baker’s yeast SSU is made up of a sin-gle 18S rRNA of 1798 nucleotides and 32 r-proteins, whereas the LSU is composed of 25S RNA of 3392 nucleotides, the 5.8S RNA of 158 nucleo-tides, the 5S RNA of 121 nucleotides (Verschoor et al. 1998) and 46 r-proteins (Smith et al. 2008).

rRNA molecules are derived from 100 to 200 copies of rDNA genes lo-calized on chromosome XII in head-to-tail manner (Venema et al. 1999). The rDNA unit-gene in yeast S. cerevisiae is 9.1 kb long. It contains a tran-scribed section that gives rise to the 35S precursor rRNA, which is processed to the mature 18S, 5,8S and 25S rRNA, and two non transcribed intergenic

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sequences (IGS). The IGSs are interjected by the coding sequence for 5S rRNA. The 35S precursor contains two external transcribed spacer (ETS) sequences. Two internal transcribed spacers (ITS) surround 5,8S rRNA pre-cursor sequence that lies in between 18S and 25S pre-rRNAs (Venema et al. 1999) and (Fig. 4).

Figure 4. Schematic representation of rDNA unit-gene. (IGS) intergenic spacer, (5S) 5S rRNA coding gene, (ETS) external transcribed spacer, (ITS) internal transcribed spacer, (18S, 5.8S and 25S) 18S, 5.8S and 25S rDNA coding sequence, respectively. (T) terminator region.

Polymerases I and III are engaged in the synthesis of the various rRNA species. RNA polymerase I transcribes the 35S precursor, whereas 5S rRNA is transcribed by RNA polymerase III. The 5S rDNA is transcribed in the opposite direction to the 35S precursor rRNA (Fig. 4). The inclusion of 5S rRNA coding sequence into the rDNA unit-gene appears to be a landmark for S. cerevisiae (Venema et al. 1999).

The rRNA folds in domains: four for the 18S rRNA and six for the 25S rRNA. 5S rRNA builds an additional complex (domain), the CP, into which it is incorporated. Even though rRNA size and primary nucleotide sequence vary among organisms, the overall folded structure, including functional sites, is highly conserved, nearly identical.

3.2.2 rRNA modifications The basic structure of an rDNA gene is similar in all eukaryote species. In budding yeast, Pol I transcribes the 18S-, 5,8S- and 25S-rRNA as one giant 35S transcript (Venema et al. 1999). Pol III transcribes 5S rRNA. Concomi-tantly with the transcription an array of snoRNPs are engaged in modifying the emerging transcript. These modifications include pseudouridylation (ψ) and 2’-O-ribose methylation, conducted by Box H/ACA and Box C/D snoRNPs, respectively (Tschochner et al. 2003). Alongside with the rRNA transcription, U3 snoRNA and 17 U3 associated factors assemble to build a large (>2 MDa) SSU processome/90S pre-ribosome particle (Granneman et al. 2004) and (Fig. 5) Sequential cleavage of the 90S pre-ribosome by endo- and exonucleases at specific sites leads to conversion of 90S pre-ribosome particle into a 43S and a 66S pre-ribosome (Tschochner et al. 2003). This cleavage often occurs before the end of the transcription of the rDNA gene (Granneman et al. 2005). The 43S pre-ribosome is exported to the cyto-plasm; where after an additional cleavage at the 3’-end of the 18S rRNA the

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pre-ribosomal particle is converted to the mature 40S subunit (Granneman et al. 2004). In contrast the 66S pre-ribosome has to undergo several additional maturation steps before the mature 60S subunit is transported from the nu-cleus to the cytoplasm (Granneman et al. 2004) (Fig. 5.)

Figure 5. Model for ribosome subunit maturation and export. Adopted from (Tschochner et al. 2003)

3.3. Ribosome assembly Ribosome biogenesis is the process in which the ribosomal particles are as-sembled and transported to the cytoplasm. In order to assure the correct as-sembly of the ribosome, its constituents have to be present in equimolar quantities. To meet this requirement, the cell mobilizes not only the expres-sion of rDNA and r-protein genes but also the expression of more than 150 auxiliary non-ribosomal components in a concerted manner (Venema et al. 1999). These include endo- and exonucleases, methyltranferases, pseu-douridine synthases, RNA chaperones, small nucleolar ribonucleoprotein particles (snoRNPs, a complex consisting of snoRNA and proteins) and RNA helicases (Tschochner et al. 2003). In eukaryotes, most of the studies concerning ribosome biogenesis have been done in S. cerevisiae.

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3.3.1 r-proteins and ribosome assembly The participation of each SSU r-protein in biogenesis of 40S ribosomes, in maturation of 18S rRNA and nuclear export of 43S pre-ribosomes, has been examined (Ferreira-Cerca et al. 2005). Thus for the SSU, almost all r-proteins belonging to this subunit, are essential for proper processing, matu-ration and cytoplasmic export of pre-SSU particles (Ferreira-Cerca et al. 2005). For instance, reduced expression of rpS30 and rpS8 led to decreased levels of 18S, but even 25S rRNA level was affected (Ferreira-Cerca et al. 2005). The SSU proteins that do not seem to be involved in pre-rRNP matu-ration are rpS7, rpS30 and rpS31 (Ferreira-Cerca et al. 2005).

In contrast, the function of most LSU r-proteins in ribosome assembly remains mostly unknown. The exceptions are rpL10, rpL11 and rpL5. RpL10 is necessary for recycling of the LSU export adaptor protein Nmd3 (West et al. 2005). Nmd3 is released from 60S particle and is recycled back to the nucleus. RpL5 and rpL11 together with Rpf2 and Rrs1 appear to be involved in the cytoplasmic export of rRNA intermediate (Zhang et al. 2007).

The assembly factors involved in recruiting r-proteins to pre-ribosomes have not been identified, except in some cases. Rrb1 is thought to play a key role in rpL3 pre-ribosome recruitment (Schaper et al. 2001) as this factor interacts with rpL3 and is implicated in its expression (Iouk et al. 2001). Similarly, Sqt1 binds to rpL10p to facilitate the incorporation of rpL10 into pre-ribosomes in the cytoplasm (West et al. 2005). Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and r-proteins rpL5 and rpL11 (Zhang et al. 2007).

Many diseases linked to ribosome biogenesis and assembly has recently been reported (Liu et al. 2006). The complexity of the described syndromes reflects the intricate mechanism of ribosome biogenesis.

3.4. Ribosome biogenesis and TOR regulation Ribosome biogenesis is of crucial importance in regulating cell growth by integrating different environmental signals. Among these are “sensing” nu-tritional status and environmental stress. Being the one of the major consum-ers of cellular energy, ribosome biogenesis has to reach a balance in produc-tion of ribosomes according to the available energy in the cell, (Fig. 6) This regulation is especially important since during ribosome biogenesis and as-sembly the cells mobilize the expression of more than 200 genes and factors (Henras et al. 2008).

Recent reports have shown that regulation of ribosome biogenesis hap-pens at the level of transcription (Li et al. 2006). This regulation involves

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Figure 6. (A) Model of TORC1 complex regulation of ribosome biogenesis (Li et al. 2006). (B) TOR regulation of RPG transcription. (Fhl1) Forkhead-like, (Ifh1) Interacting with forkhead, (CRF1) Transcriptional corepressor, (SFP1) Split Finger Protein (Transcription factor). Adopted from (Powers 2004)

modulation of activity of all the three RNA polymerases (Pol I thru III) and effects transcription regulation of genes encoding the ribosome constituents (rRNA and r-proteins) and genes encoding non-ribosomal proteins, collec-tively named Ribi for Ribosome biogenesis) (Berger et al. 2007). In budding yeast ribosome biogenesis is under control of an atypical serine/threonine kinase named TOR (target of rapamycine). TOR homologues have been identified in fungi, in mammals (mTOR) and in Drosophila (dTOR) (re-viewed in (Li et al. 2006)). In yeast two distinct proteins are involved in TOR regulation Tor1 and Tor2. Both proteins are redundant checkpoint regulators of cell growth and are sensitive to rapamycine treatment (Loewith et al. 2002). They build different complexes, TORC1 and TORC2. TORC1 contains either Tor1 or Tor2 whereas TORC2 contains only Tor2 kinase (Loewith et al. 2002). TORC1 but not TORC2 is involved in control of ribo-some biogenesis and translation. Only TORC1 complex is sensitive to rapa-

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mycine and treatment with this drug leads to rapid downregulation of genes involved in ribosome biogenesis (Li et al. 2006). The downregulation is essentially due to inhibition of Pol I, Pol II and Pol III activities.

Binding of TORC1 at the Pol I promoter on the 35S rDNA is facilitated by a helix-turn-helix motif of Tor1 (Li et al. 2006). This implicates nuclear localization of the Tor1 itself (Li et al. 2006). In addition Tor1 interacts with 5S rDNA, suggesting that Tor1 may regulate transcription of 5S rRNA by binding to the internal promoter of the 5S rDNA (Li et al. 2006).

Tor1 regulation of RPGs transcription is dependent on nuclear transport of several transcription factors (Fig. 6B). Transcription factors such as Sfp1 and co-repressor Crf1 that act on Pol II-dependent transcription are directed to the nucleus (Fig. 6B). The main transcription factors that associate with r-protein promoters are: Forkhead-like (Fhl1) and Repressor activator protein (Rap1). Fhl1 together with Interacting with forkhead (Ifh1) build a transcrip-tion complex that binds to r-protein promoters. This process is further facili-tated by Sfp1, which contributes for the full-scale transcription from r-protein promoters (Powers 2004; Hall et al. 2006) and (Fig. 6B). Tor1 regu-lates transcription of r-protein genes by maintaining the cytoplasmic local-ization of the co-repressor Crf1. Upon inhibition of Tor1 the phosphorylated form of Crf1 enters the nucleus and displaces the Ifh1 from Fhl1 complex, thus inhibiting the transcription from RPG. Phosphorylation of Crf1 is de-pendent on both Tor- and PKA dependent Yak1 kinase (Martin et al. 2004). Both Ifh1 and Crf1 share sequence homology in the domain termed Fork-head-binding (FHB) domain.

Besides TORC1 regulation of RPG transcription, other proteins might also be involved in the process of regulation of r-protein transcription. Tran-scription factors, members of the High-mobility group (HMG) are also asso-ciated with r-protein promoters via a Rap1 dependent binding. They are also linked to Pol I transcriptional activity. Though binding of the members of HMG to r-protein promoters is essential for the building Fhl1/Ifh1 complex (Berger et al. 2007) at r-protein promoters, it appears that other factors might act redundantly within the r-protein promoter context. Reports on Hmo1 binding to r-protein promoters (Hall et al. 2006) under heat shock suggest that stress conditions might have implications on RPG transcription, as well. Below I discuss the presence of heat shock elements (HSE) in the promoter of YrpL15A.

Despite the growing information on Tor-mediated regulation of ribosome biogenesis, further research is needed to unveil the subtle mechanisms of this regulation.

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4. YrpL15A

4.1. General characteristics of YrpL15A YrpL15A is a protein containing 204 amino acids (Otaka et al. 1982). The size of rpL15 is very similar in all eukaryotes and its mass has been esti-mated to approximately 24 kDa with an isoelectric point of approximately 11 (Chan et al. 1987; Tom et al. 1999; Zeng et al. 2000). YrpL15A has a high content of basic amino acids of the protein with lysine and arginine account-ing for close to 30% of the amino acids. The acidic amino acids aspartate and glutamate make up for less than 7% of the amino acids. The high pro-portion of basic amino acids probably reflects the intimate link between rpL15 and rRNA.

Eukaryotic rpL15 is homologous to rpL15e in archaebacteria (http://ribosome.miyazaki-med.ac.jp/index.html). However, there is no homologue to these proteins in eubacteria. Instead, protein rpL31 seems to replace rpL15e in the 50S subunits from eubacteria. This protein is partially homologous to rpL15e and seems to fulfil the same function in the 50S sub-unit from eubacteria (Harms et al. 2001; Klein et al. 2004). The existence of similar structural components with different sequences in biology is one expression of molecular mimicry (Smith et al. 2008).

R-protein rpL15 is one of the r-proteins that have been linked to cancer (Wang et al. 2001; Wang et al. 2006). Human rpL15 is over expressed in gastric cancer tissues as well as in gastric cancer cell lines. Inhibition of RPL15 expression by siRNA transfection, suppresses cell growth in these cancer cells (Wang et al. 2006).

4.2. YrpL15A in 60S complex Currently, there is no crystal structure of eukaryotic ribosomes that could show the precise location of rpL15A on yeast ribosomes. However, models of the yeast 60S subunit has been created based on the crystal structure of H. marismortui 50S subunit and on Cryo-EM data from yeast 60S subunit (Spahn et al. 2004). These models suggest that YrpL15A occupies a position

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in the yeast 60S subunit similar to that occupied by H. marismortui rpL15e Hmrpl15e in the 50S particle (Spahn et al. 2004).

Figure 7. Crown view of the large ribosome subunit Haloarcula marismortui (Nissen et al. 2000). (CP) central protuberance. R-protein L15e (in blue) homolog to YrpL15 and L18 (in blue) partial homolog to YrpL5

The crystal structure of Haloarcula marismortui large ribosomal subunit (Ban et al. 2000) shows, that HmrpL15e has a roughly globular domain near the surface of the ribosome and a considerable extension that goes deep into the rRNA Fig. 7 and 8. The folding of this extension is presumably com-pletely dependent on interaction with 23S rRNA. A closer analysis of the crystal structure shows that rpL15e belongs to a subgroup of r-proteins whose topography is characterized by antiparallel β-strands (Klein et al. 2004) and (Paper I, Fig. 8A). HmrpL15e is one of the proteins that bury the largest surface with rRNA and almost 60% of its available surface is in con-tact with the 23S rRNA (Ban et al. 2000; Klein et al. 2004) and (Fig. 8). An additional 11% of the protein surface is implicated in protein-protein interac-tions with Hmrp7Ae and Hmrp44e (Fig. 7). HmrpL15e makes contacts with nucleotide sequences from all domains of the 23S rRNA except domain VI, but the most extensive interaction is seen with domain I (Klein et al. 2004). This suggests that the protein may be incorporated early during ribosome assembly.

The physical proximity of HmrpL15e to PTC is depicted in Fig. 8 and de-scribed in (Smith et al. 2008).

In my studies concerning YrpL15A the efforts were concentrated on sev-eral topics. One part of the study was directed on elucidating the importance of individual amino acids and overall size of the protein on the function of

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Figure 8. Spatial proximity model of r-proteins from the large subunit to (PT) pepti-dyl transferase center in Haloarcula marismortui (Nissen et al. 2000). R-protein L15e (in green) homolog to YrpL15 and L18 (in light blue) partial homolog to YrpL5

YrpL15A. Another part was centred on the promoters of YRPL15A and the paralogous YRPL15B gene. The last part of the study concentrated on the nuclear transport and elucidation of NLS of the protein YrpL15A.

4.2.1. Mutational analysis on YrpL15A Functional implications The importance of the individual amino acids in YrpL15A has been studied by error-prone PCR technique and site-directed mutagenesis (Paper I). The results showed that YrpL15A has an unusual high tolerance to amino acid substitutions, in vivo. Thus amino acid substitutions considered to be disfa-voured were tolerated and mutant alleles containing several mutations exhib-ited a similar growth rate as the wt YrpL15A (Paper I, Supplementary table S2). This was further exemplified by the capability of rpL15B from Arabi-dopsis thaliana (ArpL15B) to functionally complement yeast wild type rpL15A (wt, YrpL15A). Despite, almost 30 % differences in amino acid sequence, ArpL15B was capable of replacing YrpL15A and resulted in a strain with growth rate similar to that of cells expressing plasmid encoded wt YrpL15A (Paper I, Fig. 6A). The observation probably shows the tremen-dous plasticity in YrpL15A structure in connection to the intimate relation-ship with surrounding rRNA, where the protein structure might be formed under the influence of the interactions with rRNA. In contrast, the more closely related r-protein L15A from Schizosaccharomyces pombe (PrpL15A) was not capable to functionally substitute YrpL15A. One explanation for this might be the two-amino acid indel at position 187 in PrpL15A. This deletion

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could result in a displacement of important rRNA binding residues. The in-ability of S. pombe PrpL15A to functionally substitute authentic YrpL15A was not due to lack of nuclear import. PrpL15A were able to enter both the nucleus and the nucleolus (Paper I Fig. 2).

In contrast, deletions and point insertions in amino acid sequence of YrpL15A were not tolerated equally well. Smaller deletions of 3-4 amino acids in the protein termini were tolerated. However, larger deletions en-compassing the 5 N-terminal and 11 C-terminal amino acids were proven deleterious for YrpL15A function (Paper I, Table 2). Point insertion result-ing in frame shift was also unviable (Paper I, Table 2). Based on the crystal structure of H. marismortui and the existent sequence homology between HmrpL15e and YrpL15A in the case of 5 N-amino acids deletion there is a possibility of interference of binding of YrpL15A to another essential r-protein YrpL8, rather than structural impairment of YrpL15A function. Point insertion resulting in frame-shift and protein with extended C-terminal was also proven unviable

4.3. Paralogues of L15 genes in yeast and promoter functions In yeast S. cerevisiae, the essential gene YRPL15A codes for the protein YrpL15A. Yeast cells also contain a second non-essential YRPL15B gene. The nucleotide sequences of these two genes differ at several positions but at the amino acid level there are only two differences. Glutamine 11 and aspar-tate 153 in YrpL15A are replaced by glutamate and asparagine, respectively in YrpL15B. These differences, in amino acid sequence between the two gene products, had no effect on the viability of the genomic knock-out strain IS1 (Paper I, Fig. 3, allele M1). YrpL15B expressed under Gal promoter could fully complement the missing genomic YRPL15A. Conversely, cells retaining the genomic copy of the YRPL15B were not able to survive dele-tion of the YRPL15A gene.

My studies presented in Paper I showed that the intra-cellular levels of mRNA coding for YrpL15A were easily detectable. In contrast, mRNA con-tent for YrpL15B was not detectable with standard RT-PCR technique, de-spite the fact that the cells tested have two copies of the YRPL15B gene but only one copy of YRPL15A gene. The result of the experiment suggested different promoter functions upstream each of the genes. In Paper I, I was able to show that the presumptive promoter region PB upstream YRPL15B was inactive. In contrast the corresponding region PA, upstream of the gene coding for YrpL15A, was functional (Paper I, Fig. 1). Reciprocal exchanges of the presumptive promoter sequences fused to the respective RPG, gave identical results. PA fused to sequence coding for YrpL15B was able to pro-

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duce functional copies of YrpL15, whereas PB fused to the sequence coding for YrpL15A was not able to sustain growth on Glucose containing medium (Paper I, Fig. 4B). Close examination for presumptive transcription factor binding sites of the upstream regions of the both genes further strengthened the experimental observations. Examination of PB upstream of YRPL15B revealed no binding site for known transcription factors. This observation is in accordance with the results in Paper I that showed that the PB promoter sequence is inactive under normal growth conditions. In contrast the region upstream of YRPL15A showed binding sites for transcription factors Rap1p and Fhl1p which are readily associated with r-protein promoters (Lascaris et al. 1999; Martin et al. 2004) and (Paper I, Fig. 4C). Another interesting fea-ture present in the upstream region of YRPL15A gene was the presence of heat shock binding elements (HSE) at 665 bp upstream of the ATG codon. The consensus sequence, of three consecutive (GAA) nucleotide repeats in PA, exhibits a build-up of a perfect type HSE described in (Yamamoto et al. 2005; Hashikawa et al. 2007). The HSE are widely distributed throughout the eukaryotic genomes and heat shock proteins have been implicated into a variety of processes like heat-induced transcription, protein degradation and detoxification (Feder et al. 1999). In yeast around 3% of the genes are poten-tial targets of Hsf1, the yeast homologue to mammalian heat-shock factors (Hahn et al. 2004). Hsf1 is constitutively bound to HSE as a homotrimer, maintaining transcription under normal growth conditions (Hashikawa et al. 2004; Hashikawa et al. 2007). Temperature fluctuations lead to elevated levels of transcription directed by Hsf1 (Erkine et al. 1999; Hahn et al. 2004). The importance of the described HSE in the upstream region of YRPL15A as well as in other r-proteins for r-protein transcription remains to be elucidated.

The physiological meaning of RPGs duplication in yeast is presently not known. One possible explanation of RPG duplication could be dose adjust-ment of the product. Another is the possibility of generation of ribosomes with slightly different properties (Komili et al. 2007). This possibility would open for a tremendous ribosomal versatility where a mixture of functionally different ribosomes would meet the need at different environmental condi-tions. Despite highly similar coding sequences, r-protein paralogues have very different untranslated regions (Komili et al. 2007). Moreover, given the example with rpL15 paralogues, the putative promoter sequences upstream each of the genes showed none marked homology. Thus, it is possible that different r-protein promoters respond to different subset of transcription factors. In line with this reasoning, duplicate RPGs in yeast and plants might result in divergence of expression of r-protein paralogues. Yet another possi-bility of the potential functions of the r-protein paralogues could be addi-tional extra-ribosomal tasks assigned to specific paralogues (Wool 1996; Warner et al. 2009).

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In contrast, in mammals only one gene is coding for each r-protein. One exception in this is the example of human rpS4 where paralogs of the r-protein are found on both X and Y chromosomes (Uechi et al. 2001).

4.4. YrpL15A and NLS In Manuscript II I studied the NLS of YrpL15A. My interest for studying the NLS of YrpL15 was raised by the hypothesis presented by Stuger (Stuger et al. 2000) that YrpL15A contained several putative NLS (pNLS) based on sequence homology to known NLS. In this study, YrpL15A was assigned three different pNLSs. In Manuscript II, I show experimentally that the NLS in YrpL15A resides in the C-terminal part. The NLS sequence includes amino acids 168GKKSRGINKGHKFNNTKA185. Orthologs of r-protein L15 from Arabidopsis thaliana and Schizosaccharomyces pombe showed similar pattern of localization as the YrpL15A (Manuscript II, Fig. 3), suggesting that both orthologs contain a NLS that could be recognized by yeast nuclear transport machinery. The identified NLS adhered to the proposed structure of a monopartite NLS (Timmers et al. 1999). Some peculiarities of the high-lighted NLS, that distinguish the sequence in YrpL15A from the NLS se-quence data presented by Chelsky (Chelsky et al. 1989) and Timmers (Timmers et al. 1999), have to be mentioned. The discovered NLS contains additional amino acids to the accepted consensus of a monopartite NLS. For clarity, I divided the discovered NLS in three parts. The first part represented by motif A (Manuscript II, Fig. 4) shows the most conserved region of the NLS that conforms to the proposed consensus sequence of a monopartite NLS. Despite its homology to a monopartite signal, Motif A was not able to direct gfp to the nucleus (Manuscript II, Fig 2, construct C5). Instead, the following less conserved motifs B and C (Manuscript II, Fig. 4) have to be present in order for the NLS to complete its nuclear targeting competence as gfp fusion construct. The inability of Motif A to direct gfp to the cell nucleus was not likely due to inability of Motif A to fold properly. Every construct presented in the study had an additional 16 amino acids spacer sequence preceding the gfp coding sequence. The discovered NLS of YrpL15A has also the capability of localizing not only to the nucleus but also to the nu-cleolus. The inbuilt capacity of an NLS to direct the transported molecule to the nucleolus has been reported in several instances (Quaye et al. 1996; Timmers et al. 1999; Kundu-Michalik et al. 2008). In the case of YrpL15A, we could observe that the nucleolar localization signal was integrated and dependent on the existing NLS.

Interestingly, despite nuclear localization of the gfp containing YrpL15A, the chimerical construct failed to incorporate in the cytoplasmic ribosomes. Similar was the case of YrpL15A tagged with V5-His6 epitope (Manuscript II, Fig. 5). Generally the newly synthesized r-proteins are rapidly transported

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in the place of ribosome assembly in the nucleolus in excess amounts (Lam et al. 2007). Following transportation into the nucleus, the r-proteins become involved in binding to the respective rRNA sequences. R-proteins that fail to incorporate in ribosomes are targeted for degradation by nuclear proteasome (Lam et al. 2007). Presence of a nuclear proteasome complex has previously been reported (Stavreva et al. 2006; Lam et al. 2007). Ubiquitination is a general and essential process by which the cell regulates the levels of pro-teins (Brooks et al. 2004). Similarly, r-proteins coupled with ubiquitin (Chan et al. 1995) show that one possible mechanism of control and degradation could ultimately been carried out by the nuclear ubiquitin-proteasome sys-tem (Lam et al. 2007). All proteins that are produced in excess are targeted for degradation. In our experiments the tagged versions of YrpL15A, despite nuclear and nucleolar localization, could not be detected on cytoplasmic ribosomes. Therefore, proteasome degradation in the nucleus could be one possible explanation for this observation.

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5. YrpL5

5.1. General characteristics of the protein YrpL5 is an essential protein for viability. In budding yeast one essential gene on chromosome XVI is coding for YrpL5. The protein is composed of 297 amino acids. In contrast to most r-proteins YrpL5 exhibits slightly acidic properties indicated by the higher ratio of acidic over basic amino acids thus containing fewer amounts of basic lysine and arginine (Nazar et al. 1979; Tang et al. 1991; Deshmukh et al. 1993). YrpL5 forms a stable complex with 5S rRNA. Similar structural complex with 5S rRNA is also observed in ribosomes from eubacteria where the 5S rRNA interacts with three r-proteins. In archaebacteria 5S rRNA interacts with two r-proteins. Some of the bacterial 5S rRNA binding proteins exhibit partial homology with rpL5, indicating that during the course of evolution a possible gene fusion has taken place (Nazar et al. 1979).

5.2 YrpL5 on 60S The position of YrpL5 on the LSU has been derived from docking the atomic model of H. marismortui 50S LSU (Ban et al. 2000) with Cryo-EM map of yeast ribosomes (Spahn et al. 2001). YrpL5 appears as a constituent of the central protuberance (CP) on LSU (Fig. 7). It makes direct contacts with YrpL11 and indirect contact to YrpL11 through 5S rRNA. Additional contact is made with expansion segment 12 (ES12), which runs on the back of the CP. ES12 is thought to induce conformational changes in the elbow regions of A- and P- site bound tRNA (Spahn et al. 2001). Additional inter-actions between the CP (LSU) and SSU in the complete 80S ribosome in-volve the intersubunit bridge B1b that is composed of YrpS18 and YrpL11 (Spahn et al. 2001). The location of YrpL5 can be deduced from H. maris-mortui HmrpL18 ortholog to YrpL5, (Fig. 7).

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5.3. L5•5S rRNA complex The initial transcription of 5S rRNA in eukaryots is started by transcription factor TFIIIA. TFIIIA is a zinc finger containing protein with affinity for both 5S rDNA and 5S rRNA itself. TFIIIA forms a 7S RNP complex with 5S rRNA. This complex is subsequently exported to the cytoplasm (Wischnewski et al. 2004). The 5S rRNA component of the 7S RNP particle interacts with the nascent YrpL5 during translation, (Fig. 9). This co-translational interaction results in the displacement of TFIIIA from 5S rRNA and formation of a stable binary complex, rpL5•5S rRNA. The nuclear re-entry of the complex is dependent on the NLS of rpL5 and is facilitated by the cellular transport receptors of the karyopherin family of proteins (Jakel et al. 1998). In the nucleus, the binary complex is recruited to the 90S pre-ribosome particle by assembly factors Rpf2 and Rrs1 (Zhang et al. 2007). Some additional proteins rpL10 and rpL11 are also recruited to rpL5•5S rRNA. Incorporation of the binary complex is necessary for further matura-tion of pre-rRNA (Zhang et al. 2007). Saturation mutagenesis studies have revealed the tremendous resilience of 5S rRNA to mutations throughout almost the whole length of yeast 5S rRNA (Smith et al. 2001). Some of the mutations increased the frameshifting of the ribosomes (Dinman et al. 1995). It is thought that 5S rRNA is capable of transmitting information between both subunits of the ribosome (Smith et al. 2001). Being the major constitu-ent of the central protuberance (CP) the binary complex rpL5•5S rRNA may present an important link between the SSU and LSU. rpL5•5S rRNA com-plex has been shown to be implicated in maintenance of translational fidel-ity. As a consequence of this, mutations in YrpL5 resulted in increased frame shifting and reduced affinities of the altered ribosomes to peptidyl-tRNA binding (Meskauskas et al. 2001). This interaction is indirect and in-volves YrpL11 and 5S rRNA as a molecular bridge (Meskauskas et al. 2001).

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Figure 9. Schematic representation of rpL5 life cycle. (ICR) internal control region, (TF) transcription factor.

5.4. YrpL5 function In my studies, Paper III and Manuscript IV, I concentrated on elucidating the importance of the N- and C-terminal sequences of YrpL5. The life cycle of rpL5 is to be intimately linked to 5S rRNA with which it forms a stable bi-nary complex (Deshmukh et al. 1993) and (Fig. 10). In yeast, extra-ribosomal 5S rRNA was also found in complex with YrpL5, indicating that YrpL5 also associates with 5S rRNA before incorporation into pre-ribosomes (Deshmukh et al. 1993). It appears that an intracellular mecha-

35

nism regulates the levels of expression of YrpL5, keeping it constant (Deshmukh et al. 1993). Upon binding, 5S rRNA and YrpL5 undergo sub-stantial conformational changes. Structurally rpL5 is proposed to contain two 5S rRNA binding regions situated in both N- (Lin et al. 2001) and C-terminal domains (Deshmukh et al. 1995). Homology studies show that the N-terminal domain is more conserved among species (Manuscript IV, Fig. 1C). It is proposed that the initial interaction between 5S rRNA and rpL5 requires amino acids 1-93, (Michael et al. 1996; Lin et al. 2001). The latter study claims that the region encompassing amino acids 35-50 is the actual region involved in 5S rRNA binding, in vitro. This binding region alone is not sufficient for complex formation (Lin et al. 2001). In addition amino acids in the C-terminal domain have importance for the 5S rRNA binding as deletions in the C-terminal region result in lethal phenotype (Nazar et al. 1982; Yaguchi et al. 1984; Deshmukh et al. 1995; Yeh et al. 1995). It is proposed that the N-terminal region, which is rich in basic amino acids, is responsible for the specific binding to 5S rRNA. The C-terminal part of the protein is thought to give an additional binding stability even if this interac-tion appears to be unspecific (Nazar et al. 1979). Yet another study has shown that 5S rRNA binding motifs in HurpL5 are localized to both the N- and the C-terminal domains and involves amino acids 1-38 and 252-297, respectively (Rosorius et al. 2000).

The C-terminal part of YrpL5 has been proposed to build an α-helix en-compassing amino acids 260-297 (Yeh et al. 1995). In this helix several basic amino acids are observed (K262, K270, K271, K276, K279, R282, R285 and K289). The three last amino acids are proposed to lie on the same side of the helix, thus making a 5S rRNA binding surface (Yeh et al. 1995). In our stud-ies Paper III we examined the possibility of displacing K289 from the putative 5S rRNA binding surface. Our results show that the length of the YrpL5 C-terminal sequence downstream the putative α-helix is of limited importance as appending acidic amino acids or a 41 amino acids C-terminal tag gave functional proteins, capable of replacing the missing genomic copy of YRPL5 in strain HMAY1 (Paper III, Table 2). Furthermore, indel A283/284 (construct YA11 in Paper III) resulted in non-functional phenotype even though the actual α-helix was retained (Paper III, Fig. 5). In this construct R285 and K289 were shifted to the opposite side of the helix, while K270, K271 and R282 positions remained intact. Double indel A283/284 and A287/288, con-struct YA7, displaces R285 and resulted in non-functional phenotype. The result suggests an important role for the R285 YrpL5 function as all functional constructs tested have R285 at the correct position (Paper III, Fig. 5). In con-trast, mutants resulting in displacement of K289 on the opposite side of the putative 5S rRNA binding surface retained functional YrpL5, (Paper III, Fig. 5, mutant YA10). This suggests that the positioning of K289 is of limited importance for the YrpL5 function in contradiction to previous reports (Yeh et al. 1995). All functional constructs YA4, YA5, YA10 and YA12 exhibited

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increased doubling time due to decreased ribosome content (Paper III, Table 2A). In the case of YA12 the increased doubling time was assigned to in-creased cell death (Paper III, Table 2A). The inability of the non-functional mutants to substitute for the missing gene YRPL5 was not due to abolished nuclear localization, as all the constructs tested were able to localize to the nucleus and to the nucleolus (Paper III, Fig. 4). The observation indicates that rpL5•5S rRNA complex in these constructs is intact. However, con-structs with displaced R285 failed to incorporate in the cytoplasmic ri-bosomes, which is in line to the data presented in (Nazar et al. 1979; Yeh et al. 1995). The results in Paper III can be summarized as follows: The length of the suggested C-terminal α-helix is of limited importance. Positioning of R285 but not of K289 is important for the proposed 5S rRNA binding. In our study we cannot rule out the possibility of existence of C-terminal α-helix. However, it is possible that other motifs in YrpL5, apart from the proposed C-terminal helix might be equally important for the correct function of YrpL5.

Figure 10. Model of rpL5•5S rRNA complex. RpL5 (helices and sheet diagram) and 5S rRNA (wire diagram)

In paper IV our focus was turned to the N-terminal part of YrpL5. Based on the protein domain organization for human rpL5 (HurpL5) presented in (Rosorius et al. 2000), we further examined the architecture of the N-terminal part in YrpL5. Our results confirmed the observations made in the article about the functional significance of amino acids 1-93. Deleting or substituting the first 18 amino acids resulted in viable constructs (Manuscript IV, Fig. 3A and 4, constructs N1 and N11). Mutants N11 and N13 showed

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also that the first 18 amino acids in the N-terminal region of YrpL5 was of limited importance for the ability of building the 5S rRNA•rpL5 complex, in contrast to the HurpL5 (Rosorius et al. 2000). Both constructs tested were viable but N13 exhibited increased doubling time at 30°C and failed to grow above this temperature (Manuscript IV, Fig. 5). Moreover, we could show that amino acids 25-66 in ArpL5 were interchangeable with YrpL5 (Manu-script IV, Fig. 4, Constructs N3, N4, N7-N9 and N13). This observation might point to the existence of a universal rRNA binding domain in both YrpL5 and ArpL5. However, further extension of amino acids from ArpL5 to include the whole region 25-90 resulted in unviable phenotype (Manu-script IV, Fig. 4, Construct N5). This latter observation might mean that species-specific sequences in ArpL5 might include the region from amino acid 67 to 90. However this assumption needs further investigation.

5.5. Nuclear transport of rpL5•5S rRNA After synthesis in the cytoplasm, all r-proteins are eventually imported in the cell nucleus. In this respect YrpL5 is a protein that exhibit the “normal” pat-tern of localization for an r-protein. In a study by (Rosorius et al. 2000) on human rpL5 (HurpL5) the domain morphology, with respect of nuclear lo-calization has been elucidated. According to this article, HurpL5 has two NLS signals. NLS1 encompasses sequence between amino acids 21-37 and NLS2 lies in between amino acids 255-297. Both NLS signals have the ca-pability of directing HurpL5 to both nucleus and nucleolus and are involved in 5S rRNA binding (Rosorius et al. 2000). A central domain containing amino acids 101-111 exhibits an accumulation of five leucines involved in the export of the protein from the nucleus to the cytoplasm, nuclear export signal (NES) (Rosorius et al. 2000). The NES region might play a role in the export of the HurpL5 protein in complex with nucleophosmin (Yu et al. 2006). One peculiar feature of HurpL5 was that the protein was imported more slowly than the other r-proteins (Lam et al. 2007).

In our studies concerning YrpL5 (manuscript IV, Fig. 2A) we could ver-ify the nuclear localization of YrpL5 to the cell nucleus and nucleolus. Three orthologs to YrpL5 from Drosophila melanogaster (DrpL5), Mus musculus (MrpL5) and Arabidopsis thaliana (ArpL5) were also tested for nuclear lo-calization in yeast S. Cerevisiae (Manuscript IV, Fig. 2A). Surprisingly ArpL5 could not localize to yeast nucleus. This observation could either point to failure in rpL5•5S rRNA complex, or disturbed nuclear transport of the latter. All other orthologs were transported to the nucleus and were even incorporated in the cytoplasmic ribosomes. Closer examination of the N-terminal part of YrpL5 (Manuscript IV, Fig. 1C) shows that a sequence in-volving 20FRRRREG26 conforms to the consensus sequence of a monopartite NLS described previously in this study. This region is well conserved among

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studied species, but one amino acid exchange is present in ArpL5 where Glutamate 25 is exchanged to Aspartate.

5.6. Dominant negative effects of non-functional YrpL5 alleles on cell growth My studies on the N- and C-terminal part of YrpL5 produced some mutants with dominant negative features when co-expressed with the wt YrpL5. There are several possible explanations to the dominant negative effects seen with the non-functional forms of YrpL5. As the binary complex L5•5S rRNA has to be incorporated in the maturing 90S particle, the unviable mu-tants of YrpL5 might compete with the existing pool of 5S rRNA producing non-functional ribosomes. If the intracellular levels of YrpL5 are under strict control (Deshmukh et al. 1993), the co-expression of the mutated and wt YrpL5 might further bias the building of non-functional ribosomes as the mutated YrpL5 is overexpressed from the Gal promoter. The possibility of the mutant forms of YrpL5 to compete for the transport factors that recruit the binary complex to the nucleus were tested with fusions between gfp and YrpL5 (Paper III, Fig. 4 and Manuscript IV, Fig. 6). The result showed that some of the non-functional constructs were directed to the nucleus thus eliminating the possibility of disturbed nuclear transport in them. Other con-structs were not directed to the nucleus and might have contributed to re-duced levels of the nuclear transport factors in the cell. Some of the mutants were detected on the cytoplasmic ribosomes. Taken together these data sug-gest for the presence of quality sensing feature in the nucleus, that “takes a decision” for the metabolic fate of r-proteins. If so, this observation can give some information on the discriminative ability of this quality checking mechanism.

The mutant constructs that were detected on the cytoplasmic ribosomes might simply mirror the fact that these mutants have less-disturbed C-termini. Nevertheless, they produce non-functional ribosomes and thus com-pete for a portion of the other ribosome constituents.

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6. Discussion

In this part of the thesis I make a comparative analysis on differences in structural and functional features of r-proteins L15A and L5 that likely stem from the different positions and roles of both r-proteins in the ribosome.

6.1. The role of the C-terminal tag Appending tags to a gene product is commonplace in molecular biology as this procedure further facilitates study and detection of the genes of interest. In my studies focused on rpL15A and rpL5 I used C-terminal V5-His6 or gfp tags to detect and follow both proteins in the cells. RpL15A and rpL5 oc-cupy different positions on yeast ribosome and play different physiological roles. Consequently, extending their C-terminal sequences resulted in differ-ent physiological implications. Probably due to its tight association with the rRNA core structure, YrpL15A lost its functionality when the C-terminus was extended with a V5-His6 tag or with a gfp tag (Paper I, Fig. 2A). The additional amino acid extensions did not interfere with the nuclear import of the protein. YrpL15A with C-terminal extensions were detected in the cell nucleus and in the nucleolus (Figure 3A, Paper I and Figure 2, Manuscript II).

However, the extended constructs were not incorporated into 60S sub-units that were exported to the nucleus (Paper I, Fig. 2B). Instead, all the expressed YrpL15A tagged with V5-His6 was predominantly located to the nucleus (Manuscript II, Fig. 5). These observations suggest that the YrpL15A with a C-terminal tag was not incorporated into the 60S particle due to lack of room to accommodate the C-terminal tag (Paper I, Figure 8A). In contrast to rpL15A, YrpL5 constructs containing a C-terminal V5-His6 tag was incorporated into functional 60S subunits that were exported to the cytoplasm. These functional differences between YrpL5 and YrpL15A are probably due to the different rate of association of the proteins with rRNA.

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6.3. Sensitivity to mutations and organism orthologs YrpL15A and YrpL5 showed different tolerance to amino acid exchanges in their primary sequence. The tolerance to alterations of the amino acid code was high in rpL15A and multiple amino acid exchanges were tolerated, re-sulting in proteins that could stimulate cell growth to the same extent as wild-type YrpL15A at different temperatures.

In contrast, YrpL5 had less tolerance to amino acid exchanges. Generally, alterations in the amino acid sequence resulted in increased doubling time and temperature sensitivity of cells expressing the mutant proteins, com-pared to expression of the wild type YrpL5. The diverging pattern of toler-ance to amino acid exchanges in the two r-proteins was further exemplified by the ability of YrpL15A and YrpL5 orthologs to substitute for the wild type function of the respective proteins. Surprisingly, the ortholog ArpL15B could substitute YrpL15A in yeast, whereas none of the tested orthologs of YrpL5 could substitute the native protein. These observations are difficult to explain without a proper experimental support. One possible explanation could be the physiological relationship between the r-proteins and their re-spective rRNA binding partner. In the case of rpL5, studies (Smith et al. 2001) on 5S rRNA showed that the majority of the nucleotides could be mutated without a noticeable effect on ribosome function. This could mean that the 5S rRNA tertiary structure is dependent on its interacting partner, rpL5. In contrast, the ability of rpL15A to accommodate several amino acid alterations and the ability of ArpL15B to substitute YrpL15A in vivo, might point instead to rRNA binding partners’ determinative role for rpL15 fold.

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7. Acknowledgement

Past years as PhD student was one of the grater challenges in my life. There are many people who contributed to the successful ending of this period. I would like to express my gratitude to the following people: My supervisor Professor Odd Nygård for accepting me as PhD and for all the support and help during the past years. All the former members in ONY group (Galina, Gunnar, Hossein, Rose-Marie, Lovisa, Birgit, Marika). Galina, for all uplifting discussions and for teaching me how to work hard. For our shared passion in fairy tales and Poirot. Alex for giving me hand in that crucial moment when I felt lost. My friends and colleagues at Södertörn Yann and Annelie. Without you guys nothing would have been the same. You cheered me up whenever I needed your emotional support. Thank you! Alex and Fergal for the good times in the pubs and supporting words. Fergal, for comments and suggestions in manuscripts. Håkan, Ambjörn and Yann for your nice dinners and conversations! Yann, for proofreading my thesis. Professor Inger Porsch-Hällström for support and good pranks. The administrative personnel at Södertörns högskola. Without your support I wouldn’t have managed my PhD. Ulrika, for all the discussions and good cookies. All the nice colleagues at Södertörns högskola. All Bulgarian friends for our nice parties and lively discussions. Especially Oggi, Sasho (Alex), Tanya, Volodya, Nina, Ruslan, Megi, Valja, Vjara and Lucho and all the children. Special thanks to my neighbours Radko and Sofia for the nice time spent together. Radko for taking me out so often on our shopping trips that refilled my fridge and for all pike and perch we caught at Lida. You are simply the best! Lasse for stretching your hand to me when I was desperate. Thank you! I hope you would forgive me for not calling you so long! Dario, for the nice walks, music and hospitality. My family in Bulgaria (mother, father and my brother’s family) for your everlasting support and faith in me. Life is very strange sometimes, but the bonds are unbreakable! I am still yours “little Ive”.

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My childhood friends in Bulgaria Pepi and Martin, for nice memories and friendship. Special thanks to my Mariela (My little “Nuncho”) for all the help, support and love during those crucial moments of writing my articles! Without you, nothing would have been possible!

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