Human Ribosomal Protein Research Articles

Mutations Preventing the Phosphorylation of Human Ribosomal Protein uS15 at Y38 and S48 Reduce the Efficiency of its Transfer into the Nucleolus

The ribosomal protein uS15 is one of the key proteins forming the small (40S) ribosomal subunit structure around the central domain of 18S rRNA. According to a number of proteomic studies based on mass-spectrometry analysis, there are many phosphorylation sites in this protein. However, when the protein uS15 is contained in the 40S subunit, it does not carry such posttranslational modification. In this study, it was found that the cytoplasmic protein kinases of HEK293T cells are able to phosphorylate the human recombinant ribosomal protein uS15 with significantly greater efficiency than that of the nucleus protein kinases. The effect of the amino acid substitutions Y38A and S48A preventing the phosphorylation of the ribosomal protein uS15 at the corresponding sites, on the transport of the recombinant protein uS15 fused to the green fluorescent protein in the nucleolus was studied. It was shown that single mutations at the above sites have little effect on the transport of this protein, whereas double mutation reduces the efficiency of this process by more than a quarter. The findings suggest the importance of phosphorylation of the ribosomal protein uS15 by cytoplasmic protein kinases at several sites, including Y38A and S48A, for its efficient transfer into the nucleolus, where pre-ribosomal subunits are assembled.

Exploring the Interaction of Human Ribosomal Protein uS3 with Single-Stranded DNAs Having Different Sequences

The analysis of data on genomic DNA sequences in the binding sites of human ribosomal protein uS3 in vivo, obtained earlier by ChIP-Seq method, was carried out; the presence of significant content of TGGAA repeats, belonging to the type III of human satellites, was found. The dependence of the affinity of the corresponding recombinant protein uS3 to single-stranded DNA and its AP lyase activity on the DNA sequence in vitro, in particular on the presence of TGGAA pentamer repeats, was investigated. It was shown that the presence of these repeats in the model DNAs did not provide an increased affinity to the isolated ribosomal protein uS3. The presence of TCC and TTC motifs in DNA complementary to the corresponding pentamer triplets only slightly increased the affinity of DNA to this protein. It was established that the AP lyase activity of the uS3 protein was also not increased when DNA containing abasic (AP) site contained TGGAA repeats and was independent on the position of the AP site in it. It was shown that the efficiency of single-stranded DNA cleavage at the AP site as a whole correlated with the affinity of uS3 protein to this DNA. It was concluded that the high content of the above repeats in the ribosomal protein uS3 binding sites on chromatin relate to high content of (TGGAA)n repeats in single-stranded DNA regions physically accessible for the protein binding but not to its higher affinity to the pentamer or DNA motifs complementary to it.

Ribosomal protein uL3 targets E2F1 and Cyclin D1 in cancer cell response to nucleolar stress

Several experimental strategies in the treatment of cancer include drug alteration of cell cycle regulatory pathways as a useful strategy. Extra-ribosomal functions of human ribosomal protein L3 (uL3) may affect DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrated that uL3 is required for the activation of G1/S transition genes. Luciferase assays established that uL3 negatively regulates the activity of E2F1 promoter. Induced ribosome-free uL3 reduces Cyclin D1 mRNA and protein levels. Using protein/protein immunoprecipitation methods, we demonstrated that uL3 physically interacts with PARP-1 affecting E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by uL3 involving E2F1 and Cyclin D1 in the regulation of cell cycle progression.

In silico evidence of de novo interactions between ribosomal and Epstein - Barr virus proteins

BackgroundAssociation of Epstein-Barr virus (EBV) encoded latent gene products with host ribosomal proteins (RPs) has not been fully explored, despite their involvement in the aetiology of several human cancers. To gain an insight into their plausible interactions, we employed a computational approach that encompasses structural alignment, gene ontology analysis, pathway analysis, and molecular docking.ResultsIn this study, the alignment analysis based on structural similarity allows the prediction of 48 potential interactions between 27 human RPs and the EBV proteins EBNA1, LMP1, LMP2A, and LMP2B. Gene ontology analysis of the putative protein-protein interactions (PPIs) reveals their probable involvement in RNA binding, ribosome biogenesis, metabolic and biosynthetic processes, and gene regulation. Pathway analysis shows their possible participation in viral infection strategies (viral translation), as well as oncogenesis (Wnt and EGFR signalling pathways). Finally, our molecular docking assay predicts the functional interactions of EBNA1 with four RPs individually: EBNA1-eS10, EBNA1-eS25, EBNA1-uL10 and EBNA1-uL11.ConclusionThese interactions have never been revealed previously via either experimental or in silico approach. We envisage that the calculated interactions between the ribosomal and EBV proteins herein would provide a hypothetical model for future experimental studies on the functional relationship between ribosomal proteins and EBV infection.

Tetrapeptide 60–63 of human ribosomal protein uS3 is crucial for translation initiation

Conserved ribosomal protein uS3 contains a decapeptide fragment in positions 55–64 (human numbering), which has a very specific ability to cross-link to various RNA derivatives bearing aldehyde groups, likely provided by K62. It has been shown that during translation in the cell-free protein-synthesizing system, uS3 becomes accessible for such cross-linking only after eIF3j leaves the mRNA binding channel of the 40S ribosomal subunit. We studied the functional role of K62 and its nearest neighbors in the ribosomal assembly and translation with the use of HEK293T-derived cell cultures capable of producing FLAG-tagged uS3 (uS3FLAG) or its mutant form with amino acid residues at positions 60–63 replaced with alanines. Analysis of polysome profiles from the respective cells and cytosol lysates showed that the mutation significantly affected the uS3 ability to participate in the assembly of 40S subunits, but it was not essential for their maturation and did not prevent the binding of mRNAs to 40S subunits during translation initiation. The most striking effect of the replacement of amino acid residues in the above uS3 positions was that it almost completely deprived the 40S subunits of their ability to form 80S ribosomes, suggesting that the 48S pre-initiation complexes assembled on these subunits were defective in the binding of 60S subunits. Thus, our results revealed the previously unknown crucial role of the uS3 tetrapeptide 60GEKG63 in translation initiation related to maintaining the proper structure of the 48S complex, most likely via the prevention of premature mRNA loading into the ribosomal channel.

An improved method for the heterologous production of soluble human ribosomal proteins in Escherichia coli

Human ribosomal proteins play important structural and functional roles in the ribosome and in protein synthesis. An efficient method to recombinantly produce and purify these proteins would enable their full characterisation. However, the production of human ribosomal proteins can be challenging. The only published method about the recombinant production of human ribosomal proteins involved the recovery of proteins from inclusion bodies, a process that is tedious and may lead to significant loss of yield. Herein, we explored the use of different Escherichia coli competent cells and fusion protein tags for the recombinant production of human ribosomal proteins. We found that, by using thioredoxin as a fusion protein, soluble ribosomal protein could be obtained directly from cell lysates, thus leading to an improved method to recombinantly produce these proteins.

RNA-binding protein RPS3 contributes to hepatocarcinogenesis by post-transcriptionally up-regulating SIRT1.

Although several studies indicate that RNA-binding proteins (RBPs) contribute to key steps in a variety of physiological processes and cancer, the detailed biological functions and mechanisms remain to be determined. By performing bioinformatics analysis using well-established hepatocellular carcinoma (HCC) datasets, we identified a set of HCC progression-associated RBPs (HPARBPs) and found that the global expression of HPARBPs was significantly correlated with patient prognosis. Among the 42 HPARBPs, human ribosomal protein S3 (RPS3) was one of the most abundant genes whose role remains uncharacterized in HCC. Gain- and loss-of-function analyses demonstrated that RPS3 promoted HCC tumorigenesis both in vitro and in vivo. Mechanistically, we revealed that silent information regulator 1 (SIRT1) was a critical target of RPS3 and was essential for sustaining the RPS3-induced malignant phenotypes of HCC cells. RPS3 stabilized SIRT1 mRNA by binding to AUUUA motifs in the 3448–3530 region of the 3′ untranslated region (UTR) of SIRT1 mRNA. In addition, we found that (5-formylfuran-2-yl) methyl 4-hydroxy-2-methylenebutanoate (FMHM) inhibited HCC progression by repressing the RPS3/SIRT1 pathway. Our study unveils a novel extra-ribosomal role of RPS3 in facilitating hepatocarcinogenesis via the post-transcriptional regulation of SIRT1 expression and proposes that the RPS3/SIRT1 pathway serves as a potential therapeutic target in HCC.

Abstract IA09: RiboSeq and ye shall find: Noncanonical protein translation in cancer Iimunosurveillance

Abstract By displaying oligopeptides on the cell surface, class I major histocompatibility complex (MHC-I) molecules enable CD8+ T cell immunosurveillance of tumor cells. MHC-I peptide ligands derive proteins synthesized by cancer cells. A number of lines of evidence indicate that many MHC-I peptides derive from a pool of rapidly degraded defective ribosomal products (DRiPs). DRiPs include misfolded gene products resulting from errors in transcription, translation, folding, targeting, and assembly. They also encompass the translation products of “immunoribosomes,” specialized ribosomes proposed to generate antigenic peptides for the express propose of immunosurveillance. There are now several examples in which noncanonical translation efficiently generates peptides for immunosurveillance. I will discuss how the method of ribosome profiling can be used to identify novel tumor target peptides for immunotherapy arising from noncanonical translation. To determine whether ribosome heterogeneity itself can also selectively increase antigenic peptide generation, we screened a lentiviral shRNA library targeting each of the 80 human ribosomal protein genes. We identified candidate genes by alterations in steady state class I molecule expression (a measure of overall peptide supply) or in the ratio of source proteins to source protein-derived peptides. Two ribosomal proteins that either increase or decrease the efficiency of peptide generation are near neighbors on the ribosome surface, suggesting possible mechanisms for influencing peptide generation. Citation Format: Jonathan W. Yewdell. RiboSeq and ye shall find: Noncanonical protein translation in cancer Iimunosurveillance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA09.

Short Open Reading Frames and Their Encoded Peptides.

Proteomics 2018, 18, 1700035 DOI: 10.1002/pmic.201700035 In this article, the following sentences and reference were erroneously published: “In their technical brief, Khitun and Slavoff describe their workflow to detect and validate non-annotated proteins with non-AUG start sites.[11] This proteomic detection method is also exemplified with the characterization of the non-AUG initiated small protein (alt-RPL36) co-encoded with human ribosomal protein L36.” [11] S. A. K. Slavoff, Proteomics 2018 The Editorial Office apologizes for this error.

Immortalization of chicken embryonic liver-derived cell line by stable expression of hMRP18S-2 for serotype 4 fowl adenovirus propagation

Inclusion body hepatitis and hydropericardium-hepatitis syndrome caused by serotype 4 fowl adenovirus (FAdV-4) have emerged in China since 2013. FAdV is usually propagated in primary chicken embryonic liver cells or embryo yolk sac. The aim of this work was to develop an immortalized CEL cell line by stable expression of human mitochondrial ribosomal protein 18S-2, named CEL-hMRP18S-2 cells, for the propagation of FAdV-4. The maximum cell density of CEL-hMRP18S-2 cells could reach 2.65 × 106 cells/ml in four-days culture. According to the mRNA levels of cell-cycle related genes in CEL-hMRP18S-2 cells tested by qRT-PCR, we speculated that the transformation of hMRP18S-2 into CEL cells caused the functional inactivation of p53 and the significant down-regulation of p15INK4b might cause the hyperphosphorylated form of Rb, releasing E2F-1 factor and enhancing the E2F-dependent transcription for cell cycle progression. It was suspected that the up-regulated c-Myc mRNA level at the initial period of immortalization might prompt transformed cells through the G0-G1 checkpoint. The normal CPE was observed in CEL-hMRP18S-2 cells infected by FAdV-4 and microcarrier suspension culture performed for FAdV-4 propagation with 9.0 lgTCID50/ml suggested that CEL-hMRP18S-2 cells could be a useful continuous cell line for isolation of wild FAdV and production of FAdV-inactivated vaccine.

The eS26 protein is involved in the formation of a nucleophosmin binding site on the human 40S ribosomal subunit

Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26FLAG). The production of eS26FLAG in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change. Exogenous eS26FLAG was able to replace endogenous eS26 in 40S ribosomal subunits, without affecting the assembly and translational activity of 80S ribosomes. However, eS26FLAG-containing ribosome fractions from the respective polysome profile displayed a reduced content of nucleophosmin, a multifunctional protein, which, as is known, is involved in the formation and nuclear export of ribosomal subunits. In general, our data showed that although the appearance of the FLAG tag at the C-terminus of eS26 does not affect translation, it interferes with nucleophosmin incorporation into the 40S subunit, pointing out the importance of the C-terminus integrity of eS26 for nucleophosmin binding. In addition, with the recombinant protein, we demonstrated the binding of nucleophosmin to both isolated eS26 and 40S subunits in the presence of HeLa nuclear extract that phosphorylated the recombinant nucleophosmin. These findings suggest that for nuclear export, nucleophosmin could directly bind to pre-40S subunits in the mRNA exit site region where the C-terminus of eS26 is located.

Human ribosomal protein eS1 is engaged in cellular events related to processing and functioning of U11 snRNA.

Ribosomal proteins are involved in many cellular processes through interactions with various RNAs. Here, applying the photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation approach to HEK293 cells overproducing ribosomal protein (rp) eS1, we determined the products of RNU5A-1 and RNU11 genes encoding U5 and U11 snRNAs as the RNA partners of ribosome-unbound rp eS1. U11 pre-snRNA-associated rp eS1 was revealed in the cytoplasm and nucleus where rp eS1-bound U11/U12 di-snRNP was also found. Utilizing recombinant rp eS1 and 4-thiouridine-containing U11 snRNA transcript, we identified an N-terminal peptide contacting the U-rich sequence in the Sm site-containing RNA region. We also showed that the rp eS1 binding site on U11 snRNA is located in the cleft between stem-loops I and III and that its structure mimics the respective site on the 18S rRNA. It was found that cell depletion of rp eS1 leads to a decrease in the splicing efficiency of minor introns and to an increase in the level of U11 pre-snRNA with the unprocessed 3′ terminus. Our findings demonstrate the engagement of human rp eS1 in events related to the U11 snRNA processing and to minor-class splicing. Contacts of rp eS1 with U5 snRNA in the minor pre-catalytic spliceosome are discussed.

Rat embryonic fibroblasts immortalized by MRPS18-2 protein are target for NK-cells.

Overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts (REFs). The derived cells (18IM) expressed embryonic stem cell markers. Noteworthy, genes encoding the COX family proteins were up-regulated significantly. It is known that the COX family proteins are involved in the regulation of immune response.In the present work we demonstrate that 18IM cells behave like stem cells when subjected to directed differentiation in vitro. However, unlike stem cells, 18IM cells do not develop tumors in vivo, in SCID mice. This phenomenon is observed due to the strong natural killer (NK) cell immunogenicity. 18IM cells were better recognized by NK cells, compared with primary REFs, as was shown by a standard NK killing assay.Our data explain asymmetry in behavior of stem-like cells in vivo and in vitro, and this support the notion that stem and/or cancer-initiating cells are preferred targets for NK-cells. Concluding, the S18-2 protein is a putative target for cancer vaccines.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Role of uL3 in Multidrug Resistance in p53-Mutated Lung Cancer Cells.

Cancer is one of the most common causes of death among adults. Chemotherapy is crucial in determining patient survival and quality of life. However, the development of multidrug resistance (MDR) continues to pose a significant challenge in the management of cancer. In this study, we analyzed the role of human ribosomal protein uL3 (formerly rpL3) in multidrug resistance. Our studies revealed that uL3 is a key determinant of multidrug resistance in p53-mutated lung cancer cells by controlling the cell redox status. We established and characterized a multidrug resistant Calu-6 cell line. We found that uL3 down-regulation correlates positively with multidrug resistance. Restoration of the uL3 protein level re-sensitized the resistant cells to the drug by regulating the reactive oxygen species (ROS) levels, glutathione content, glutamate release, and cystine uptake. Chromatin immunoprecipitation experiments and luciferase assays demonstrated that uL3 coordinated the expression of stress-response genes acting as transcriptional repressors of solute carrier family 7 member 11 (xCT) and glutathione S-transferase α1 (GST-α1), independently of Nuclear factor erythroid 2-related factor 2 (Nrf2). Altogether our results describe a new function of uL3 as a regulator of oxidative stress response genes and advance our understanding of the molecular mechanisms underlying multidrug resistance in cancers.

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.

Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin.

Ricin is a type II ribosome-inactivating protein (RIP) that depurinates A4324 at the sarcin-ricin loop of 28 S ribosomal RNA (rRNA), thus inactivating the ribosome by preventing elongation factors from binding to the GTPase activation centre. Recent studies have disclosed that the conserved C-terminal domain (CTD) of eukaryotic ribosomal P stalk proteins is involved in the process that RIPs target ribosome. However, the details of the molecular interaction between ricin and P stalk proteins remain unknown. Here, we report the structure of ricin-A chain (RTA) in a complex with the CTD of the human ribosomal protein P2. The structure shows that the Phe111, Leu113 and Phe114 residues of P2 insert into a hydrophobic pocket formed by the Tyr183, Arg235, Phe240 and Ile251 residues of RTA, while Asp115 of P2 forms hydrogen bonds with Arg235 of RTA. The key residues in RTA and P2 for complex formation were mutated, and their importance was determined by pull-down assays. The results from cell-free translation assays further confirmed that the interaction with P stalk proteins is essential for the inhibition of protein synthesis by RTA. Taken together, our results provide a structural basis that will improve our understanding of the process by which ricin targets the ribosome, which will benefit the development of effective small-molecule inhibitors for use as therapeutic agents.

Patterns of ribosomal protein expression specify normal and malignant human cells.

BackgroundRibosomes are highly conserved molecular machines whose core composition has traditionally been regarded as invariant. However, recent studies have reported intriguing differences in the expression of some ribosomal proteins (RPs) across tissues and highly specific effects on the translation of individual mRNAs.ResultsTo determine whether RPs are more generally linked to cell identity, we analyze the heterogeneity of RP expression in a large set of human tissues, primary cells, and tumors. We find that about a quarter of human RPs exhibit tissue-specific expression and that primary hematopoietic cells display the most complex patterns of RP expression, likely shaped by context-restricted transcriptional regulators. Strikingly, we uncover patterns of dysregulated expression of individual RPs across cancer types that arise through copy number variations and are predictive for disease progression.ConclusionsOur study reveals an unanticipated plasticity of RP expression across normal and malignant human cell types and provides a foundation for future characterization of cellular behaviors that are orchestrated by specific RPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1104-z) contains supplementary material, which is available to authorized users.

Integrative analyses shed new light on human ribosomal protein gene regulation.

Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals.

Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress.

The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology.