Extra-ribosomal Functions Research Articles

Measuring the dynamics of E. coli ribosome biogenesis using pulse-labeling and quantitative mass spectrometry

The ribosome is an essential organelle responsible for cellular protein synthesis. Until recently, the study of ribosome assembly has been largely limited to in vitro assays, with few attempts to reconcile these results with the more complex ribosome biogenesis process inside the living cell. Here, we characterize the ribosome synthesis and assembly pathway for each of the E. coli ribosomal protein (r-protein) in vivo using a stable isotope pulse-labeling timecourse. Isotope incorporation into assembled ribosomes was measured by quantitative mass spectrometry (qMS) and fit using steady-state flux models. Most r-proteins exhibit precursor pools ranging in size from 0% to 7% of completed ribosomes, and the sizes of these individual r-protein pools correlate well with the order of r-protein binding in vitro. Additionally, we observe anomalously large precursor pools for specific r-proteins with known extra-ribosomal functions, as well as three r-proteins that apparently turnover during steady-state growth. Taken together, this highly precise, time-dependent proteomic qMS approach should prove useful in future studies of ribosome biogenesis and could be easily extended to explore other complex biological processes in a cellular context.

Dual Functions of the C5a Receptor as a Connector for the K562 Erythroblast-Like Cell-THP-1 Macrophage-Like Cell Island and as a Sensor for the Differentiation of the K562 Erythroblast-Like Cell during Haemin-Induced Erythropoiesis

The transcriptional nuclear factor binding to the Y box of human leukocyte antigen genes (NF-Y) for the C5a receptor (C5aR) gene is active in erythroblasts. However, the roles of the C5aR in erythropoiesis are unclear. We have previously demonstrated that apoptotic cell-derived ribosomal protein S19 (RP S19) oligomers exhibit extraribosomal functions in promoting monocyte chemotaxis and proapoptosis via the C5aR without receptor internalisation. In contrast to the extraribosomal functions of the RP S19, a proapoptotic signal in pro-EBs, which is caused by mutations in the RP S19 gene, is associated with the inherited erythroblastopenia, Diamond-Blackfan anaemia. In this study, we detected C5aR expression and RP S19 oligomer generation in human erythroleukemia K562 cells during haemin-induced erythropoiesis. Under monocell culture conditions, the differentiation into K562 erythrocyte-like cells was enhanced following the overexpression of Wild-type RP S19. Conversely, the differentiation was repressed following the overexpression of mutant RP S19. An RP S19 oligomer inhibitor and a C5aR inhibitor blocked the association of the K562 basophilic EB-like cells and the THP-1 macrophage-like cells under coculture conditions. When bound to RP S19 oligomers, the C5aR may exhibit dual functions as a connector for the EB-macrophage island and as a sensor for EB differentiation in the bone marrow.

Extraribosomal functions of bacterial ribosomal proteins

Though their primary role in a cell is to serve as integral components of protein synthesis machinery, the ribosome, many of them have functions beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with other cellular components. Extraribosomal activities of some ribosomal proteins have been observed as early as in the 1970-1980s. During the last years both a list of r-proteins-moonlighters and the repertoire of their additional functions beyond the ribosome have been greatly expanded, mainly due to newly developed techniques for dissecting RNA/DNA-protein or protein-protein interactions within functional complexes involved in various cellular processes. In this review, we surveyed information on the experimentally proven as well as on presumptive extraribosomal functions which may be performed by bacterial r-proteins in a cell.

SiRNA Knockdown of Ribosomal Protein Gene RPL19 Abrogates the Aggressive Phenotype of Human Prostate Cancer

We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.

Ribosomal Protein S3: A Multifunctional Target of Attaching/Effacing Bacterial Pathogens

The extraribosomal functions of ribosomal proteins have drawn significant recent attention. Ribosomal protein S3 (RPS3), a component of the eukaryotic 40S ribosomal subunit, is a multifunctional protein that regulates DNA repair, apoptosis, and the innate immune response to bacterial infection. Here we the review the latest findings about RPS3 extraribosomal functions, with special emphasis on their relation to microbial pathogenesis and enteropathogenic Escherichia coli.

Molecular characterization and structure analysis of RPL10/QM-like protein from the red drum Sciaenops ocellatus (Sciaenidae)

The QM-like gene encodes a ribosomal protein L10. Besides housekeeping roles in protein synthesis, QM-like proteins have multiple extraribosomal functions during cell growth, cell differentiation and apoptosis. We obtained the full-length cDNA of QM-like protein (designated as SoQM) from the salt water game fish Sciaenops ocellatus, using RACE-PCR. The sequence consists of 740 bp, encoding 215-amino acid residues with 24.60 kDa. The AA sequence of the SoQM protein contains a series of functional motifs that belong to the QM family signature, which is conserved among different species. The SoQM gene contains five introns and six exons. The expression pattern of SoQM as determined by RT-PCR indicated that SoQM mRNA was expressed in all tissues tested, including brain, gill, head-kidney, intestine, stomach, heart, spleen, blood, muscle, and gonads. The phylogenetic tree constructed with MEGA 4.0 showed that SoQM clusters together with that of other fish. It was found that the sequences of the SoQM gene are highly conserved, suggesting the fundamental and critical functions of SoQM in S. ocellatus. The three-dimensional structure of the SoQM protein core domain (4~169) was predicted by the Swiss-Model program. Compared with QM proteins in other species, the main structure of SoQM protein was conserved, while the C-terminal domain was different from other QM-like proteins. Prediction of the three-dimensional structure of SoQM would provide valuable insight into the molecular basis of protein function, allowing an effective design of experiments, such as site-directed mutagenesis, studies of disease-related mutations or structure-based design of specific inhibitors.

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated ‘rpL22-like short’) that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.

Arabidopsis L10 ribosomal proteins in UV-B responses

Ribosomal protein L10 (RPL10) is an ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; however, increasing evidences indicate that RPL10 from various organisms has multiple extra ribosomal functions, besides being a constituent of ribosome and its role in translation. Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C. Previously, we found that in maize and in A. thaliana, UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B. Here, we show that RPL10s are differentially regulated by UV-B in a dosage and time dependent manner: RPL10C is induced, RPL10B is down regulated at high UV-B intensity, and RPL10A is not UV-B regulated. In addition, by coimmunoprecipitation studies using RPL10 antibodies and proteins from control and UV-B irradiated Arabidopsis plants, we demonstrate that RPL10 associates with different proteins under the two different conditions, including nuclear proteins, suggesting that at least one isoform may have extra-ribosomal roles.

The nucleolus: structure/function relationship in RNA metabolism

The nucleolus is the ribosome factory of the cells. This is the nuclear domain where ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. Here we describe the classical tripartite organization of the nucleolus in mammals, reflecting ribosomal gene transcription and pre-ribosomal RNA (pre-rRNA) processing efficiency: fibrillar center, dense fibrillar component, and granular component. We review the nucleolar organization across evolution from the bipartite organization in yeast to the tripartite organization in humans. We discuss the basic principles of nucleolar assembly and nucleolar structure/function relationship in RNA metabolism. The control of nucleolar assembly is presented as well as the role of pre-existing machineries and pre-rRNAs inherited from the previous cell cycle. In addition, nucleoli carry many essential extra ribosomal functions and are closely linked to cellular homeostasis and human health. The last part of this review presents recent advances in nucleolar dysfunctions in human pathology such as cancer and virus infections that modify the nucleolar organization.

Ribosomal Protein S3, a New Substrate of Akt, Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.

Extraribosomal functions associated with the C terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G1 phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

The other lives of ribosomal proteins

Despite the fact that ribosomal proteins are the constituents of an organelle that is present in every cell, they show a surprising level of regulation, and several of them have also been shown to have other extra-ribosomal functions, such in replication, transcription, splicing or even ageing. This review provides a comprehensive summary of these important aspects.

Comprehensive molecular structure of the eukaryotic ribosome.

Despite the emergence of a large number of X-ray crystallographic models of the bacterial 70S ribosome over the past decade, an accurate atomic model of the eukaryotic 80S ribosome is still not available. Eukaryotic ribosomes possess more ribosomal proteins and ribosomal RNA than do bacterial ribosomes, which are implicated in extraribosomal functions in the eukaryotic cells. By combining cryo-EM with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome complete with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. We have localized one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans. Additionally, we characterize crucial interactions between the dynamic stalk base of the ribosome with eukaryotic elongation factor 2.

Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium

Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.

Role of large subunit ribosomal protein L4 in ribosome maturation and cell cycle progression in Saccharomyces cerevisiae

Eukaryotic ribosome biogenesis is a highly co‐coordinated multi‐step process that requires equimolar synthesis of several rRNAs and over 50 ribosomal proteins. Until recently, ribosomal proteins were mainly considered only as structural components of the ribosomes. This view has now changed with increasing evidence indicating consistent alterations in r‐protein expression levels during tumorogenesis and in certain developmental defects.Ribosomal protein L4 is a large ribosomal subunit protein that is structurally conserved in all kingdoms of life. Furthermore, there are several reports of alteration in regulation of L4 mRNA during tissue regeneration, interaction of L4 with transcriptional factors, and varied regulation of L4 mRNA in cancer cell lines. This indicates that L4 has key extra ribosomal functions along with its role in ribosomal biogenesis and function. Here we show that in vivo depletion of Rpl4p in S. cerevisiae results in severe loss of 60S ribosomal subunits. Northern blot, primer extension, and uracil pulse‐labeling experiments suggest this might be due to a block in the processing of the 27SA3 precursor RNA into 5.8S and 25S rRNA as well as a delay in processing of 35S precursor indicating an important role in maturation of the large subunit of the ribosome. More surprisingly, depletion of Rpl4 and select few large subunit ribosomal proteins results in accumulation of multiple budded phenotype. FACS analysis further confirms accumulation of 3N peak representing the multi budded phenotype. Initial microscopic analysis shows that the Rpl4p depleted cells might be getting arrested in late anaphase / early telophase with separated nuclei but disorganized micro tubules. Further experiments are being done to characterize this phenotype and to elucidate the role of Rpl4 in this particular cell cycle defect

Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.

Emerging functions of ribosomal proteins in gene-specific transcription and translation

Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-κB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

A Large Ribosomal Subunit Protein Abnormality in Diamond-Blackfan Anemia (DBA).

DBA is an inherited bone marrow failure syndrome characterized by hypoproliferative anemia, congenital abnormalities and cancer predisposition. Ribosomal genes RPS19 and 24 are mutated in 25% and 3% of DBA patients respectively. To identify additional genetic abnormalities in DBA, we evaluated 2 unrelated children with DBA and sub-telomeric deletions of chromosome 3q by comparative genomic hybridization. The larger deletion spanned 11 Mb from 3q28 to the telomeric region and included 72 gene candidates. The second deletion involved 4 Mb from 3q29 to the telomeric end and included 52 known or hypothetical genes. The overlapping deletion region contained a previously reported 1.5 Mb microdeletion-associated syndrome that did not involve hematologic abnormalities, leaving 24 candidate genes. Gene expression microarray analysis from patient-derived EBV cell lines demonstrated down regulation of 7 of these candidate genes, one of which was RPL35a, a component of the large ribosomal subunit. We screened for mutations of RPL35a by direct sequencing of PCR-amplified genomic DNA from 149 DBA probands (125 sporadic, 24 familial) and 180 normal control subjects. We identified three probands with sequence changes in the RPL35a coding region: 1) an in-frame deletion in exon 3 (82-84CTT), causing a deletion of leucine at codon 28, 2) a nonsense mutation in exon 4 (298C>T), leading to an Arg102Stop and a 9 amino acid C-terminal truncation and 3) a missense mutation in exon 3 (97G>A) leading to a Val33Ile change. In the patient derived EBV cell line, the latter sequence change also resulted in an aberrant exon 3 splice site leading to a frame shift following codon 32. All of the probands with RPL35a mutations were sporadic cases. These sequence variations were not observed in the control subjects. Four lentiviral-based siRNA constructs targeting RPL35a were used to test the functional consequences of reduced RPL35a expression. Hematopoietic cell lines (TF-1 and UT-7/epo) transduced with the RPL35a directed siRNA constructs demonstrated decreased growth and viability compared to control siRNAs. Northern blot analysis demonstrated abnormal processing of large ribosomal subunit RNA with decreased mature 5.8S and 28S as well as decreased precursor 12S and 32S rRNA. Orthophosphate labeling confirmed a kinetic defect in large subunit rRNA processing, characterized by increased amounts of 45S and 41S rRNA with decreases of the precursors to and the mature 28S and 5.8S rRNAs. Mature 18S rRNA levels were unaffected, suggesting a defect in rRNA processing within the first internal transcribed sequence (ITS1). These data demonstrate that DBA can be caused by alterations in large as well as small ribosomal subunit proteins. These observations further support the hypothesis that altered ribosome homeostasis and function, rather than extra-ribosomal gene functions, is the central mechanism leading to DBA.

Translational machinery of the chaetognath Spadella cephaloptera: a transcriptomic approach to the analysis of cytosolic ribosomal protein genes and their expression

BackgroundChaetognaths, or arrow worms, are small marine, bilaterally symmetrical metazoans. The objective of this study was to analyse ribosomal protein (RP) coding sequences from a published collection of expressed sequence tags (ESTs) from a chaetognath (Spadella cephaloptera) and to use them in phylogenetic studies.ResultsThis analysis has allowed us to determine the complete primary structures of 23 out of 32 RPs from the small ribosomal subunit (SSU) and 32 out of 47 RPs from the large ribosomal subunit (LSU). Ten proteins are partially determined and 14 proteins are missing. Phylogenetic analyses of concatenated RPs from six animals (chaetognath, echinoderm, mammalian, insect, mollusc and sponge) and one fungal taxa do not resolve the chaetognath phylogenetic position, although each mega-sequence comprises approximately 5,000 amino acid residues. This is probably due to the extremely biased base composition and to the high evolutionary rates in chaetognaths. However, the analysis of chaetognath RP genes revealed three unique features in the animal Kingdom. First, whereas generally in animals one RP appeared to have a single type of mRNA, two or more genes are generally transcribed for one RP type in chaetognath. Second, cDNAs with complete 5'-ends encoding a given protein sequence can be divided in two sub-groups according to a short region in their 5'-ends: two novel and highly conserved elements have been identified (5'-TAATTGAGTAGTTT-3' and 5'-TATTAAGTACTAC-3') which could correspond to different transcription factor binding sites on paralog RP genes. And, third, the overall number of deduced paralogous RPs is very high compared to those published for other animals.ConclusionThese results suggest that in chaetognaths the deleterious effects of the presence of paralogous RPs, such as apoptosis or cancer are avoided, and also that in each protein family, some of the members could have tissue-specific and extra-ribosomal functions. These results are congruent with the hypotheses of an allopolyploid origin of this phylum and of a ribosome heterogeneity.

Ribosomal Proteins and Colorectal Cancer

The ribosome is essential for protein synthesis. The composition and structure of ribosomes from several organisms have been determined, and it is well documented that ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) constitute this important organelle. Many RPs also fill various roles that are independent of protein biosynthesis, called extraribosomal functions. These functions include DNA replication, transcription and repair, RNA splicing and modification, cell growth and proliferation, regulation of apoptosis and development, and cellular transformation. Previous investigations have revealed that RP regulation in colorectal carcinomas (CRC) differs from that found in colorectal adenoma or normal mucosa, with some RPs being up-regulated while others are down-regulated. The expression patterns of RPs are associated with the differentiation, progression or metastasis of CRC. Additionally, the recent literature has shown that the perturbation of specific RPs may promote certain genetic diseases and tumorigenesis. Because of the implications of RPs in disease, especially malignancy, our review sought to address several questions. Why do expression levels or categories of RPs differ in different diseases, most notably in CRC? Is this a cause or consequence of the diseases? What are their possible roles in the diseases? We review the known extraribosomal functions of RPs and associated changes in colorectal cancer and attempt to clarify the possible roles of RPs in colonic malignancy.