Download Ribosomal proteins L5 and L15 Ivailo Simoff in vivo

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 process, 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 ribosomal 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 eukaryotic 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. Furthermore, both L5 and L15 are connected to various human diseases which
makes it important to elucidate their role in ribosome biogenesis and ribosome function.
By applying random- and site-directed mutagenesis, coupled with functional 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 multiple 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 rproteins 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
CP
eEF
eIF
ES
GAP
HmrpL15e
HmrpL7Ae
HurpL5
LSU
NLS
NPC
PrpL15A
PTC
RF
RPG
rpS or L
r-proteins
rRNA
RRF
snoRNA
SSU
TOR
YeEF2
YrpL15A
YrpL5
YrpL8
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A. thaliana r-protein L15B
Central protuberance of the ribosome
Eukaryotic elongation factor
Eukaryotic initiation factor
Expansion segment
GTP-ase activating protein
H. marismortui r-protein L15e
H. marismortui r-protein L7Ae
H. sapiens r-protein L5
Large subunit of the ribosome
Nuclear localization signal
Nuclear pore complex
S. pombe r-protein L15A
Peptidyl transferase centre
Release factor
r-protein coding gene
r-protein from SSU or LSU, respectively
Ribosomal proteins
Ribosomal RNA
Ribosome recycling factor
Small nucleolar RNA
Small subunit of the ribosome
Target of rapamycine
S. cerevisiae elongation factor 2
S. cerevisiae r-protein L15A
S. cerevisiae r-protein L5
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 residing in a gene is translated into a sequence of amino acids that folds into a 3dimensional protein structure. A large molecular machine called the ribosome 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, elongation 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 ribosomal 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 compartments 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 transcription a plethora of modifying enzymes, protein factors and r-proteins are engaged 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 Saccharomyces 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 microscopy (Gomez-Lorenzo et al. 2000; Spahn et al. 2001; Taylor et al. 2007).
The eukaryotic and prokaryotic ribosomes share same morphological features. 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 different 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. cerevisiae.
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 interactions sites for tRNA. The tRNA interaction sites are referred to as the aminoacyl (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 accommodates 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 transferase 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 translation regulation. This need is met by increasing the complexity in for example
the initiation process in eukaryotes (Spahn et al. 2001). Another consequence of compartmentalisation is the need for transport of proteins e.g. rproteins 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)). Binding 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 preinitiation complex with the mRNP particle leads to formation of new complex 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 particle, 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 elongation 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’ hydroxyl 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 growing peptide chain of the P-site bound peptidyl-tRNA (Met-tRNAi in the reaction 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 peptidyltRNA in the A-site and a deacylated tRNA positioned in the P-site. The peptidyl-tRNA has to be moved back to the P-site to restore the ability of the Asite to accept the next aminoacyl-tRNA. The translocation, which is facilitated by elongation factor 2 (eEF2), also involves the simultaneous movement 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 contrast, the updated model GTP hydrolysis is suggested to occur prior to ribosome 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 binding protein.
During the translation process several intermediate states of tRNA binding 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, respectively, 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 facilitated 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, codonindependent, 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 ribosome recycling factor (RRF) is present that promotes ribosome subunit dissociation. 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, rproteins 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 domain that is often exposed to the solvent side of the ribosomal subunits. The
globular domain frequently contains acidic amino acids needed for interaction with additional components (Wilson et al. 2005). The primary role of
eukaryotic r-proteins is to stabilize the interactions between different domains 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 contrast, 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 rprotein. In budding yeast, 59 of the 78 RPGs are present as doubles (Wolfe
et al. 1997). The exact physiological meaning of these duplications is presently 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 transcription 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 rproteins 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 nuclear 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 regu18
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 special 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. Association 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 concentration than in nucleus. The nucleotide state of Ran is regulated by additional factors associated, RanGTP-ase activating protein (RanGAP),
As the ribosome assembly occurs in the nucleolar compartment of the nucleus, nuclear import of r-proteins from the cytoplasm is necessary to maintain 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 importin-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 typically composed of basic amino acids K and/or R. Dependent on the structural 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) βkaryopherin, (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 dipeptide (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 single 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 nucleotides, the 5S RNA of 121 nucleotides (Verschoor et al. 1998) and 46 rproteins (Smith et al. 2008).
rRNA molecules are derived from 100 to 200 copies of rDNA genes localized 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 transcribed 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 precursor 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. Concomitantly 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 endoand 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 cytoplasm; where after an additional cleavage at the 3’-end of the 18S rRNA the
21
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 nucleus 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 assembled and transported to the cytoplasm. In order to assure the correct assembly of the ribosome, its constituents have to be present in equimolar
quantities. To meet this requirement, the cell mobilizes not only the expression 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, pseudouridine 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.
22
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 rproteins belonging to this subunit, are essential for proper processing, maturation 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 maturation 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” nutritional status and environmental stress. Being the one of the major consumers of cellular energy, ribosome biogenesis has to reach a balance in production of ribosomes according to the available energy in the cell, (Fig. 6) This
regulation is especially important since during ribosome biogenesis and assembly 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 happens at the level of transcription (Li et al. 2006). This regulation involves
23
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, collectively 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) (reviewed 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 ribosome biogenesis and translation. Only TORC1 complex is sensitive to rapa24
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 rprotein promoters are: Forkhead-like (Fhl1) and Repressor activator protein
(Rap1). Fhl1 together with Interacting with forkhead (Ifh1) build a transcription complex that binds to r-protein promoters. This process is further facilitated by Sfp1, which contributes for the full-scale transcription from rprotein promoters (Powers 2004; Hall et al. 2006) and (Fig. 6B). Tor1 regulates transcription of r-protein genes by maintaining the cytoplasmic localization 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 dependent on both Tor- and PKA dependent Yak1 kinase (Martin et al. 2004).
Both Ifh1 and Crf1 share sequence homology in the domain termed Forkhead-binding (FHB) domain.
Besides TORC1 regulation of RPG transcription, other proteins might
also be involved in the process of regulation of r-protein transcription. Transcription factors, members of the High-mobility group (HMG) are also associated 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.
25
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 estimated 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 accounting 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 proportion 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 subunit 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
26
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 completely 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 contact 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 interactions 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 described in (Smith et al. 2008).
In my studies concerning YrpL15A the efforts were concentrated on several 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
27
Figure 8. Spatial proximity model of r-proteins from the large subunit to (PT) peptidyl 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 disfavoured were tolerated and mutant alleles containing several mutations exhibited a similar growth rate as the wt YrpL15A (Paper I, Supplementary table
S2). This was further exemplified by the capability of rpL15B from Arabidopsis 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 tremendous plasticity in YrpL15A structure in connection to the intimate relationship 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
28
could result in a displacement of important rRNA binding residues. The inability 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 encompassing the 5 N-terminal and 11 C-terminal amino acids were proven
deleterious for YrpL15A function (Paper I, Table 2). Point insertion resulting 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 rprotein 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 aspartate 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 deletion 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 content for YrpL15B was not detectable with standard RT-PCR technique, despite 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 pro29
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 feature 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 potential 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 adjustment 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 conditions. 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 possibility of the potential functions of the r-protein paralogues could be additional extra-ribosomal tasks assigned to specific paralogues (Wool 1996;
Warner et al. 2009).
30
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 rprotein 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 highlighted NLS, that distinguish the sequence in YrpL15A from the NLS sequence 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 nucleolus. 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
31
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 proteins (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 system (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.
32
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 rproteins. 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 interactions between the CP (LSU) and SSU in the complete 80S ribosome involve the intersubunit bridge B1b that is composed of YrpS18 and YrpL11
(Spahn et al. 2001). The location of YrpL5 can be deduced from H. marismortui HmrpL18 ortholog to YrpL5, (Fig. 7).
33
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 cotranslational interaction results in the displacement of TFIIIA from 5S rRNA
and formation of a stable binary complex, rpL5•5S rRNA. The nuclear reentry 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 preribosome 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 maturation 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 constituent of the central protuberance (CP) the binary complex rpL5•5S rRNA may
present an important link between the SSU and LSU. rpL5•5S rRNA complex has been shown to be implicated in maintenance of translational fidelity. As a consequence of this, mutations in YrpL5 resulted in increased
frame shifting and reduced affinities of the altered ribosomes to peptidyltRNA binding (Meskauskas et al. 2001). This interaction is indirect and involves YrpL11 and 5S rRNA as a molecular bridge (Meskauskas et al.
2001).
34
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 binary complex (Deshmukh et al. 1993) and (Fig. 10). In yeast, extraribosomal 5S rRNA was also found in complex with YrpL5, indicating that
YrpL5 also associates with 5S rRNA before incorporation into preribosomes (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 substantial conformational changes. Structurally rpL5 is proposed to contain
two 5S rRNA binding regions situated in both N- (Lin et al. 2001) and Cterminal 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 interaction 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 Nand 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 encompassing 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 studies 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 Cterminal 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, construct 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 contrast, 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
36
increased doubling time due to decreased ribosome content (Paper III, Table
2A). In the case of YA12 the increased doubling time was assigned to increased 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, constructs with displaced R285 failed to incorporate in the cytoplasmic ribosomes, 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 Nterminal 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
37
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 (Manuscript 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 (Manuscript 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” pattern 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 localization 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 capability 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 verify 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 localization 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 Nterminal part of YrpL5 (Manuscript IV, Fig. 1C) shows that a sequence involving 20FRRRREG26 conforms to the consensus sequence of a monopartite
NLS described previously in this study. This region is well conserved among
38
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 mutants 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 constructs were not directed to the nucleus and might have contributed to reduced levels of the nuclear transport factors in the cell. Some of the mutants
were detected on the cytoplasmic ribosomes. Taken together these data suggest 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 Ctermini. Nevertheless, they produce non-functional ribosomes and thus compete for a portion of the other ribosome constituents.
39
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 occupy different positions on yeast ribosome and play different physiological
roles. Consequently, extending their C-terminal sequences resulted in different 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 subunits 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.
40
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, resulting 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, compared to expression of the wild type YrpL5. The diverging pattern of tolerance 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 respective 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.
41
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, RoseMarie, 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”.
42
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!
43
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