Showing papers on "Ribosomal protein published in 2006"

Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases

Abstract: The target of rapamycin (TOR) signal-transduction pathway is an important mechanism by which eucaryotic cells adjust their protein biosynthetic capacity to nutrient availability. Both in yeast and in mammals, the TOR pathway regulates the synthesis of ribosomal components, including transcription and processing of pre-rRNA, expression of ribosomal proteins and the synthesis of 5S rRNA. Expression of the genes encoding the numerous constituents of ribosomes requires transcription by all three classes of nuclear RNA polymerases. In this review, we summarize recent advances in understanding the interplay among nutrient availability, transcriptional control and ribosome biogenesis. We focus on transcription in response to nutrients, detailing the relevant downstream targets of TOR in yeast and mammals. The critical role of TOR in linking environmental queues to ribosome biogenesis provides an efficient means by which cells alter their overall protein biosynthetic capacity.

Nutrient regulates Tor1 nuclear localization and association with rDNA promoter

Abstract: Yeast Tor1 is dynamically distributed in the cytoplasm and nucleus, and its association with the rDNA promoter is important for 35S rRNA synthesis and cell growth. TOR is the target of the immunosuppressant rapamycin and a key regulator of cell growth. It modulates diverse cellular processes in the cytoplasm and nucleus1,2,3,4,5, including the expression of amino acid transporters, ribosomal RNAs and ribosomal proteins. Despite considerable recent progress, little is known about the spatial and temporal regulation of TOR signalling, particularly that leading into the nucleus. Here we show that Tor1 is dynamically distributed in the cytoplasm and nucleus in yeast. Tor1 nuclear localization is nutrient dependent and rapamycin sensitive: starvation or treatment with rapamycin causes Tor1 to exit from the nucleus. Tor1 nuclear localization is critical for 35S rRNA synthesis, but not for the expression of amino acid transporters and ribosomal protein genes. We show further that Tor1 is associated with 35S ribosomal DNA (rDNA) promoter chromatin in a rapamycin- and starvation-sensitive manner; this association is necessary for 35S rRNA synthesis and cell growth. These results indicate that the spatial regulation of TOR complex 1 (TORC1) might be involved in differential control of its target genes. TOR is known as a classic cytoplasmic kinase that mediates the cytoplasm-to-nucleus signalling by controlling the localization of transcription factors. Our data indicate that TOR might be more intimately involved in gene regulation than previously thought.

Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes.

Abstract: Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

Ribosomal Protein S24 Gene Is Mutated in Diamond-Blackfan Anemia

Abstract: Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit

Abstract: A newly identified step in maturation of the small ribosomal particle regulated by the kinase Hrr25 is critical, for without Hrr25, immature 40S subunits accumulate. The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments1,2,3,4. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed5,6,7,8,9,10,11,12. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the ‘beak’. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Abstract: Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CC AUGG, in which the purines at −3 and +4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon–anticodon base-pairing during 48S initiation complex formation, and that the role of −3 and +4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at +4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C1696 and less well to AA1818–1819 in helix 44 of 18S rRNA, G cross-linked exclusively to AA1818–1819. U at −3 cross-linked to rpS5 and eIF2α, whereas G cross-linked only to eIF2α. Results of UV cross-linking experiments and of assays of 48S complex formation done using α-subunit-deficient eIF2 indicate that eIF2α’s interaction with the −3 purine is responsible for recognition of the −3 context position by 43S complexes and suggest that the +4 purine/AA1818–1819 interaction might be responsible for recognizing the +4 position.

Nucleophosmin is essential for ribosomal protein L5 nuclear export.

Abstract: Nucleophosmin (NPM/B23) is a key regulator in the regulation of a number of processes including centrosome duplication, maintenance of genomic integrity, and ribosome biogenesis While the mechanisms underlying NPM function are largely uncharacterized, NPM loss results in severe dysregulation of developmental and growth-related events We show that NPM utilizes a conserved CRM1-dependent nuclear export sequence in its amino terminus to enable its shuttling between the nucleolus/nucleus and cytoplasm In search of NPM trafficking targets, we biochemically purified NPM-bound protein complexes from HeLa cell lysates Consistent with NPM's proposed role in ribosome biogenesis, we isolated ribosomal protein L5 (rpL5), a known chaperone for the 5S rRNA Direct interaction of NPM with rpL5 mediated the colocalization of NPM with maturing nuclear 60S ribosomal subunits, as well as newly exported and assembled 80S ribosomes and polysomes Inhibition of NPM shuttling or loss of NPM blocked the nuclear export of rpL5 and 5S rRNA, resulting in cell cycle arrest and demonstrating that NPM and its nuclear export provide a unique and necessary chaperoning activity to rpL5/5S

RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea.

Abstract: The trace element selenium is found in proteins as selenocysteine (Sec), the 21st amino acid to participate in ribosome-mediated translation. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. Its biosynthesis from seryl-tRNASec has been established for bacteria, but the mechanism of conversion from Ser-tRNASec remained unresolved for archaea and eukarya. Here, we provide evidence for a different route present in these domains of life that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNASec kinase (PSTK) converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS); this protein was previously annotated as SLA/LP. The human and archaeal SepSecS genes complement in vivo an Escherichia coli Sec synthase (SelA) deletion strain. Furthermore, purified recombinant SepSecS converts Sep-tRNASec into Sec-tRNASec in vitro in the presence of sodium selenite and purified recombinant E. coli selenophosphate synthetase (SelD). Phylogenetic arguments suggest that Sec decoding was present in the last universal common ancestor. SepSecS and PSTK coevolved with the archaeal and eukaryotic lineages, but the history of PSTK is marked by several horizontal gene transfer events, including transfer to non-Sec-decoding Cyanobacteria and fungi.

Nucleolus: from structure to dynamics

Abstract: The nucleolus, a large nuclear domain, is the ribosome factory of the cells. Ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins in the nucleolus, and the ribosome subunits are then transported to the cytoplasm. In this review, the structural organization of the nucleolus and the dynamics of the nucleolar proteins are discussed in an attempt to link both information. By electron microscopy, three main nucleolar components corresponding to different steps of ribosome biogenesis are identified and the nucleolar organization reflects its activity. Time-lapse videomicroscopy and fluorescent recovery after photobleaching (FRAP) demonstrate that mobility of GFP-tagged nucleolar proteins is slower in the nucleolus than in the nucleoplasm. Fluorescent recovery rates change with inhibition of transcription, decreased temperature and depletion of ATP, indicating that recovery is correlated with cell activity. At the exit of mitosis, the nucleolar processing machinery is first concentrated in prenucleolar bodies (PNBs). The dynamics of the PNBs suggests a steady state favoring residence of processing factors that are then released in a control- and time-dependent manner. Time-lapse analysis of fluorescence resonance energy transfer demonstrates that processing complexes are formed in PNBs. Finally, the nucleolus appears at the center of several trafficking pathways in the nucleus.

Dendritic mRNAs encode diversified functionalities in hippocampal pyramidal neurons.

Abstract: Targeted transport of messenger RNA and local protein synthesis near the synapse are important for synaptic plasticity. In order to gain an overview of the composition of the dendritic mRNA pool, we dissected out stratum radiatum (dendritic lamina) from rat hippocampal CA1 region and compared its mRNA content with that of stratum pyramidale (cell body layer) using a set of cDNA microarrays. RNAs that have over-representation in the dendritic fraction were annotated and sorted into function groups. We have identified 154 dendritic mRNA candidates, which can be arranged into the categories of receptors and channels, signaling molecules, cytoskeleton and adhesion molecules, and factors that are involved in membrane trafficking, in protein synthesis, in posttranslational protein modification, and in protein degradation. Previously known dendritic mRNAs such as MAP2, calmodulin, and G protein gamma subunit were identified from our screening, as were mRNAs that encode proteins known to be important for synaptic plasticity and memory, such as spinophilin, Pumilio, eEF1A, and MHC class I molecules. Furthermore, mRNAs coding for ribosomal proteins were also found in dendrites. Our results suggest that neurons transport a variety of mRNAs to dendrites, not only those directly involved in modulating synaptic plasticity, but also others that play more common roles in cellular metabolism.

An HMG protein, Hmo1, associates with promoters of many ribosomal protein genes and throughout the rRNA gene locus in Saccharomyces cerevisiae.

Abstract: HMG proteins are architectural proteins that bind to DNA with low sequence specificity, but little is known about their genomic location and biological functions. Saccharomyces cerevisiae encodes 10 HMG proteins, including Hmo1, which is important for maximal transcription of rRNA. Here we use chromatin immunoprecipitation coupled with microarray analysis to determine the genome-wide association of Hmo1. Unexpectedly, Hmo1 binds strongly to the promoters of most ribosomal protein (RP) genes and to a number of other specific genomic locations. Hmo1 binding to RP promoters requires Rap1 and (to a lesser extent) Fhl1, proteins that also associate with RP promoters. Hmo1, like Fhl1 and Ifh1, typically associates with an IFHL motif in RP promoters, but deletion of the IFHL motif has a very modest effect on Hmo1 binding. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity. These results suggest that Hmo1 is required for the assembly of transcription factor complexes containing Fhl1 and Ifh1 at RP promoters and that proteins other than Fhl1 and Ifh1 also play an important role in RP transcription. Lastly, like mammalian UBF, Hmo1 associates at many locations throughout the rRNA gene locus, and it is important for processing of rRNA in addition to its role in rRNA transcription. We speculate that Hmo1 has a role in coordinating the transcription of rRNA and RP genes.

MDMX regulation of p53 response to ribosomal stress

Abstract: Ribosomal stress such as disruption of rRNA biogenesis activates p53 by release of ribosomal proteins from the nucleoli, which bind to MDM2 and inhibit p53 degradation. We found that p53 activation by ribosomal stress requires degradation of MDMX in an MDM2-dependent fashion. Tumor cells overexpressing MDMX are less sensitive to actinomycin D-induced growth arrest due to formation of inactive p53–MDMX complexes. Knockdown of MDMX increases sensitivity to actinomycin D, whereas MDMX overexpression abrogates p53 activation and prevents growth arrest. Furthermore, MDMX expression promotes resistance to the chemotherapeutic agent 5-fluorouracil (5-FU), which at low concentrations activates p53 by inducing ribosomal stress without significant DNA damage signaling. Knockdown of MDMX abrogates HCT116 tumor xenograft formation in nude mice. MDMX overexpression does not accelerate tumor growth but increases resistance to 5-FU treatment in vivo. Therefore, MDMX is an important regulator of p53 response to ribosomal stress and RNA-targeting chemotherapy agents.

Formation of nsP3-Specific Protein Complexes during Sindbis Virus Replication

Abstract: Alphaviruses are arthropod-borne viruses (arboviruses) that include a number of important human and animal pathogens. Their replication proceeds in the cytoplasm of infected cells and does not directly depend on nuclei. Alphaviruses encode only four nonstructural proteins that are required for the replication of viral genome and transcription of the subgenomic RNA. However, the replicative enzyme complexes (RCs) appear to include cellular proteins and assemble on cellular organelles. We have developed a set of recombinant Sindbis (SIN) viruses with green fluorescent protein (GFP) insertions in one of the nonstructural proteins, nsP3, to further understand the RCs' genesis and structure. We studied the assembly of nsP3/GFP-containing protein complexes at different stages of infection and isolated a combination of cellular proteins that are associated with SIN nsP3. We demonstrated the following. (i) SIN nsP3 can tolerate the insertion of GFP into different fragments of the coding sequence; the designed recombinant viruses are viable, and their replication leads to the assembly of nsP3/GFP chimeric proteins into gradually developing, higher-order structures differently organized at early and late times postinfection. (ii) At late times postinfection, nsP3 is assembled into complexes of similar sizes, which appear to be bound to cytoskeleton filaments and can aggregate into larger structures. (iii) Protein complexes that are associated with nsP3/GFP contain a high concentration of cytoskeleton proteins, chaperones, elongation factor 1A, heterogeneous nuclear ribonucleoproteins, 14-3-3 proteins, and some of the ribosomal proteins. These proteins are proposed to be essential for SIN RC formation and/or functioning.

Tobacco plastid ribosomal protein S18 is essential for cell survival

Abstract: Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Deltarps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.

The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly

Abstract: The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

Abstract: Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.

Fine-structure analysis of ribosomal protein gene transcription.

Abstract: The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.

Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm.

Abstract: Eleven reference genes (18s ribosomal ribonucleic acid [RNA], 28s ribosomal RNA, ubiquitin, beta-actin, glycerine aldehyde dehydrogenase, ATP-synthase subunit 5B, hydroxymethyl-bilane synthase, hypoxanthine-phosphoribosyl transferase, ribosomal protein L32, tryptophan 5-monooxygenase activation protein (zeta polypeptide), and TATA-Box binding protein) were analyzed in use as references for gene expression profiling experiments using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in canine mammary tumors. The transcription level of the candidates was measured in 22 histologically characterized excised tumor specimens from mammary gland tissue and 22 samples of non-neoplastic mammary tissue samples from the same individuals. Results were used to rank candidate reference genes using the GeNorm tool. It was determined that in samples of canine mammary gland tissue, a combination of hypoxanthine-phosphoribosyl transferase, ATP-synthase subunit 5B, ribosomal protein L32 and ubiquitin yields stable reference gene expression levels, whereas the use of glycerin aldehyde dehydrogenase or ribosomal RNA is unsuitable for normalization of qRT-PCR results in this tissue type.

The budding yeast rRNA and ribosome biosynthesis (RRB) regulon contains over 200 genes

Abstract: The ribosome biogenesis pathway constitutes one of the major metabolic obligations for a dividing yeast cell and it depends upon the activity of hundreds of gene products to produce the necessary rRNA and ribosomal protein components. Previously, we reported that a set of 65 S. cerevisiae genes that function in the rRNA biosynthesis pathway are transcriptionally co-regulated as cells pass through a variety of physiological transitions. By analysing multiple microarray-based transcriptional datasets, we have extended that study and now suggest that the ribosomal and rRNA biosynthesis regulon contains over 200 genes. This regulon is distinct from the set of ribosomal protein genes, and the promoters of the expanded RRB gene set are highly enriched for the PAC and RRPE motifs. Since a similar pattern of organization and gene regulation can be recognized in C. albicans, the RRB regulon appears to be a conserved, extensive, and metabolically important group of genes.

TOPs and their regulation

Abstract: Upon cell-cycle arrest or nutrient deprivation, the cellular rate of ribosome production is reduced significantly. In mammalian cells, this effect is achieved in part through a co-ordinated inhibition of RP (ribosomal protein) synthesis. More specifically, translation initiation on RP mRNAs is inhibited. Translational regulation of RP synthesis is dependent on cis -elements within the 5′-UTRs (5′-untranslated regions) of the RP mRNAs. In particular, a highly conserved 5′-TOP (5′-terminal oligopyrimidine tract) appears to play a key role in the regulation of RP mRNA translation. This article explores recent developments in our understanding of the mechanism of TOP mRNA regulation, focusing on upstream signalling pathways and trans -acting factors, and highlighting some interesting observations which have come to light following the recent development of cDNA microarray technology coupled with polysome analysis.

Overexpression of ribosomal protein L15 is associated with cell proliferation in gastric cancer

Abstract: Ribosomal proteins are the components of ribosome, which also exhibit various secondary functions in DNA repair, apoptosis, drug resistance and proliferation. In our previous study of microarray, ribosomal protein L15 (RPL15) was identified as an upregulated gene in gastric cancer. We investigated the expression of ribosomal protein L15 in gastric cancer and the effect of RPL15 on proliferation of gastric cancer. It was found that the expression of RPL15 was markedly up-regulated in gastric cancer tissues. RPL15 was also highly expressed in gastric cancer cell lines AGS, MKN45, MKN28, SGC7901 and KATOIII. Inhibition of RPL15 expression by siRNA vector transfection suppressed the growth of SGC7901 cells significantly, which was independent of the expression of Cyclin D1 and B1. Down-regulation of RPL15 expression inhibited SGC7901 cell growth in soft agar and its tumorigenicity in nude mice. RPL15 promotes cell proliferation and may be a potential target for anticancer therapy of gastric cancer.

Ltv1 Is Required for Efficient Nuclear Export of the Ribosomal Small Subunit in Saccharomyces cerevisiae

Abstract: In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Deltaltv1 mutants, as judged by the altered distribution of the 5'-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.

Drosophila ribosomal proteins are associated with linker histone H1 and suppress gene transcription

Abstract: The dynamics and function of ribosomal proteins in the cell nucleus remain enigmatic. Here we provide evidence that specific components of Drosophila melanogaster ribosomes copurify with linker histone H1. Using various experimental approaches, we demonstrate that this association of nuclear ribosomal proteins with histone H1 is specific, and that colocalization occurs on condensed chromatin in vivo. Chromatin immunoprecipitation analysis confirmed that specific ribosomal proteins are associated with chromatin in a histone H1-dependent manner. Overexpression of either histone H1 or ribosomal protein L22 in Drosophila cells resulted in global suppression of the same set of genes, while depletion of H1 and L22 caused up-regulation of tested genes, suggesting that H1 and ribosomal proteins are essential for transcriptional gene repression. Overall, this study provides evidence for a previously undefined link between ribosomal proteins and chromatin, and suggests a role for this association in transcriptional regulation in higher eukaryotes.

The essential GTPase RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis.

Abstract: Summary In this paper the essential GTPase YlqF is shown to participate in the biogenesis of the 50S ribosomal subunit in Bacillus subtilis . Cells depleted of YlqF displayed gene expression profiles and nucleoid morphologies that were consistent with a function for YlqF in translation. In addition, YlqF is evolutionarily linked to two eukaryotic GTPases, Nog2p and Nug1p, that are involved in the biogenesis and the nuclear export of the 60S ribosomal subunit. Analysis of ribosomes from cells depleted of YlqF demonstrated that the formation of 70S ribosomes was greatly reduced and the large subunit sedimented at 45S. Cells grown with varying depleted levels of YlqF, yielding doubling times ranging from 38 min to 150 min, all displayed the 45S intermediate. Purified YlqF-His 6 protein associates with the 45S intermediate, but not the mature 50S subunit in vitro . Analysis of proteins from the 45S intermediate indicated that ribosomal protein L16, which is added late during in vitro Escherichia coli 50S ribosome biogenesis, was missing from the 45S intermediate. These results support a model in which YlqF participates in the formation of active 70S ribosomes in the cell by functioning in a late step of 50S subunit biogenesis. Based on these results we propose to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).