Ribosomal protein uS3/RPS3 in eukaryotes is an essential component of the 40S ribosomal subunit, where it interacts with mRNA and initiation factors. It also has various extra-ribosomal functions. Proteomic studies have identified several ubiquitination sites in human uS3 that could potentially serve either as degradation signals to limit the protein level or as regulatory signals to target uS3 into cellular pathways. Currently, only uS3 ubiquitination at K214 is well understood. Here, we obtain information on functional assignment of uS3 ubiquitination at K62 and K230, using HEK293T cells producing FLAG-tagged uS3 or its forms with replacements K62R or K230R incapable of ubiquitination. Both mutations lead to a large increase of the total uS3 level in the cell indicating the involvement of the respective sites in a degradatory ubiquitination. A comparison of the level of reporter mRNAs in the total cell lysate and in the polysomal fractions revealed that both K62 and K230 are involved in the regulation of the cellular level of ribosome-free uS3 and their replacement with arginine reduces translation efficiency. Furthermore, free uS3 increases the relative levels of translated mRNA of NF-κB-dependent genes such as IL8, IL23A, and CD40, supporting its involvement in the NF-κB pathway.

INTRODUCTION:Exosomes, extracellular vesicles (EVs) 30-150 nm in diameter, are known to mediate intercellular communication by transferring bioactive molecules that can reprogram recipient cell phenotypes. Our analysis of 5-year survival data from breast cancer patients (BCPs) revealed that the poorest outcomes were exclusively associated with conserved signature of three ribosomal proteins (RPs) - MRPL52, RPL28, and RPL40 (UBA52) - that were completely absent in healthy females (HFs) and better-prognosis cases. Strikingly, all patients with this exosomal RP profile succumbed to metastatic relapse within the observation period. METHODS:While RPs are known components of tumor-derived exosomes, their absence in EVs from normal cells suggests cancer-specific sorting mechanisms. To investigate this, we characterized exosomal RP cargo from three breast cancer cell lines (MCF-7, BT-474, BT-549), finding cell-type-specific RP patterns that differed from the clinical BC signature. Transfection of BT-549 cells with FLAG-tagged RPS3 (an aggressive cancer-associated RP) induced dramatic changes in exosomal RP composition, including the appearance of RPL28 - matching our clinical findings. RESULTS:Bioinformatic analysis revealed that the RPs identified in BCP blood exosomes (MRPL52, RPL28, RPL40, RPS6) are associated with poor prognosis across multiple cancers and participate in key oncogenic pathways. CONCLUSION:The concordance between our clinical and experimental data suggests that: (a) specific RPs are selectively packaged into exosomes under stress conditions; (b) this process may contribute to BC aggressiveness; (c) exosomal RP profiling could serve as a novel prognostic approach. These findings illuminate previously unrecognized roles of ribosomal components in cancer progression through EV-mediated communication.

Protein synthesis is a fundamental biological process universally mediated by ribosomes—complex ribonucleoprotein assemblies responsible for translating genetic information into functional proteins. Despite significant structural information provided by X-ray crystallography and cryo-electron microscopy (cryo-EM), certain dynamic features of ribosomal function, particularly those involving RNA conformational flexibility and transient interactions, remain challenging to characterize. Electron Paramagnetic Resonance (EPR) spectroscopy, combined with site-directed spin labeling (SDSL), has emerged as a robust complementary approach for probing structural dynamics and conformational heterogeneity in ribosomal complexes. This review summarizes recent advances in applying EPR spectroscopy, particularly pulse dipolar EPR (DEER/PELDOR), to investigate human ribosomal complexes. We discuss methodological aspects of spin-labeling strategies for mRNA, comparing various nitroxide-based labels and highlighting their specific advantages for probing ribosomal interactions. Through representative examples, we illustrate how different EPR techniques yield complementary structural information in studying ribosome-RNA interactions. Key findings include the identification of alternative mRNA conformations within ribosomal complexes, characterization of labile RNA binding sites near the mRNA entry channel, and elucidation of stabilization effects mediated by tRNA interactions. Furthermore, we demonstrate how the integration of EPR data with molecular modeling facilitates accurate interpretation of distance distributions, enabling the correlation of experimental findings with atomic-level structural models. Finally, we address current methodological limitations of EPR spectroscopy, outlining promising perspectives and anticipated advancements in this evolving field.

In this study, we investigated the structural variability of mRNA in the human ribosome by comparing two spin-labeling strategies: one involving an mRNA analog bearing two spin labels attached to the ribose-phosphate backbone and the other placing labels at the nucleotide bases. The use of two strategies of spin labeling of mRNAs allowed us to study for the first time the effect of the structure and location of spin labels on the measured interspin distances in human ribosome complexes. Experiments using dipolar EPR spectroscopy, supported by molecular dynamics calculations, demonstrated that labels introduced at nucleotide bases provide a higher resolution between mRNA conformations in the ribosome mRNA channel, compared to labels introduced at the ribose-phosphate backbone. Although ribose-phosphate labeling turned out to be less informative on its own for studying mRNA conformations in the ribosome than the previously used base labeling, it can find application in other complex studies of the structure of RNAs and their complexes.

Many ribosomal proteins (RPs) have functions beyond their canonical role as constituents of the ribosome. They often relate to human pathologies, primarily, to carcinogenesis, and the expression of specific RPs is considerably changed in malignant cells. On the other hand, extracellular vesicles (including exosomes), which provide intercellular communication by transporting specific molecular cargo from donor to recipient cells, often contain specific sets of RPs. Thus, one can assume that oncogenic properties of RPs can be transferred from one cell to another by exosomes. Such kind transfer has been already documented with RPS3 and gastric cancer cells. However, it remains largely unclear how widespread is the above effect and to which extent it contributes to the tumor progression and metastasis. To shed light on this issue, a comparative analysis of the sets of RPs found in exosomes and of the available data on oncogenic properties of these proteins is conducted.

Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.

Ribosomal proteins (RPs), the constituents of the ribosome, belong to the most abundant proteins in the cell. A highly coordinated network of interactions implicating RPs and ribosomal RNAs (rRNAs) forms the functionally competent structure of the ribosome, enabling it to perform translation, the synthesis of polypeptide chain on the messenger RNA (mRNA) template. Several RPs contact ribosomal ligands, namely, those with transfer RNAs (tRNAs), mRNA or translation factors in the course of translation, and the contribution of a number of these particular contacts to the translation process has recently been established. Many ribosomal proteins also have various extra-ribosomal functions unrelated to translation. The least-understood and -discussed functions of RPs are those related to their participation in the intercellular communication via extracellular vesicles including exosomes, etc., which often carry RPs as passengers. Recently reported data show that such a kind of communication can reprogram a receptor cell and change its phenotype, which is associated with cancer progression and metastasis. Here, we review the state-of-art ideas on the implications of specific amino acid residues of RPs in the particular stages of the translation process in higher eukaryotes and currently available data on the transport of RPs by extracellular vesicles and its biological effects.

The human protein uS3, a component of the small ribosomal subunit, has a long-known extra-ribosomal activity as an enzyme of base excision DNA repair displayed in its ability to cleave DNA at abasic (AP) sites. It has been found that the efficacy of DNA cleavage by uS3 in vitro depends on the DNA sequence. To clarify the issue on the sequence specificity of uS3 as an AP lyase in general, we applied a combinatorial approach based on the use of a model single-stranded circular DNA with an AP site flanked with random trinucleotides at both sides. The cleavage of this DNA by uS3 under conditions when only its minor portion undergoes the reaction resulted in the formation of the linear DNA with random triplets at the 5' and 3' termini. NGS sequencing of the DNA library derived from this DNA allowed identifying the contexts within which uS3 cleaves DNA the most and the least effectively. Given that the AP lyase reaction occurs via the formation of a covalent intermediate (Schiff base), we determined the region comprising the active center of the uS3 protein. By digesting of uS3 cross-linked to a radiolabeled AP site-containing model DNA with specific proteolytic agents followed by analysis of the resulting modified oligopeptides, the cross-link was mapped to the region 155-192 (likely, to R173/R178). Thus, our results clarified two previously unstudied features of the uS3 AP lyase activity, one related to the recognition of sequences in DNA surrounding the AP site, and the other to the protein region directly contacting this site.

The protein eS26 is a structural component of the eukaryotic small ribosomal subunit involved in the formation of the mRNA binding channel in the region of the exit site. By applying site-directed cross-linking to mammalian 80S ribosomes, it has been shown that the same mRNA nucleotide residues are implicated in the interaction with both eS26 and translation initiation factor 3 (eIF3) and that contacts of the protein with mRNAs are mediated by its eukaryote-specific motif YxxPKxYxK. To examine the role of eS26 in translation, we transfected HEK293T cells with plasmid constructs encoding the wild-type FLAG-labeled protein (wt-eS26FLAG) or its forms with either a single substitution of any conserved amino acid residue in the above motif, or a simultaneous replacement of all the five ones (5A). The western blot analysis of fractions of polysome profiles from the transfected cells revealed no effects of the single mutations in eS26, but showed that the replacement of the five conserved residues led to the increased share of the light polysome fraction compared to that detected with control, wt-eS26FLAG-producing cells. In addition, the above fraction exhibited the enhanced content of the eIF3e subunit that is known to promote selective translation. These findings, together with real-time PCR data on the relative contents of specific mRNAs in light and heavy polysomes from cells producing the mutant 5A compared to those from control cells, suggest a possible involvement of the YxxPKxYxK motif of eS26 in the fine regulation of translation to maintain the required balance of synthesized proteins.

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin–spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

Abasic (AP) sites in mRNAs are lesions whose accumulation in cells is linked to various neurodegenerative diseases arising from the appearance of truncated peptides due to the premature cessation of translation of these mRNAs. It is believed that the translation of AP site-containing mRNAs is stopped when the damaged codon arrives to the A site, where it is not decoded. We propose an alternative translation arrest mechanism mediated by the 40S ribosomal subunit protein uS3. Recently, it has been shown that in human 80S ribosomal complexes assembled without translation factors, uS3 cross-links to the AP site at the 3'-terminus of the mRNA, whose undamaged part is bound at the 40S subunit channel, via its peptide 55-64 exposed near the mRNA entry pore. In this study, we examined whether such cross-linking occurs during the translation of mRNA with the AP site. To this end, we used a set of synthetic mRNAs bearing the AP site inserted in the desired location in their sequences. An analysis of 80S ribosomal complexes formed with these mRNAs in a mammalian cell-free protein-synthesizing system demonstrates that AP sites do indeed cross-link to uS3 in the course of the translation. We also show that the cross-linking occurs as soon as the AP site arrives to a common favorable position relative to uS3, which is independent on its location in the mRNA. Our findings suggest that the mechanism of stopping translation of damaged mRNAs involving uS3, along with the one mentioned above, could underlie ribosome-associated mRNA quality control.

Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases.

The conserved ribosomal protein uS3 in eukaryotes has long been known as one of the essential components of the small (40S) ribosomal subunit, which is involved in the structure of the 40S mRNA entry pore, ensuring the functioning of the 40S subunit during translation initiation. Besides, uS3, being outside the ribosome, is engaged in various cellular processes related to DNA repair, NF-kB signaling pathway and regulation of apoptosis. This review is devoted to recent data opening new horizons in understanding the roles of uS3 in such processes as the assembly and maturation of 40S subunits, ensuring proper structure of 48S pre-initiation complexes, regulation of initiation and ribosome-based RNA quality control pathways. Besides, we summarize novel results on the participation of the protein in processes beyond translation and consider biomedical implications of previously known and recently found extra-ribosomal functions of uS3, primarily, in oncogenesis.

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.

During the current decade, data on the post-translational hydroxylation of specific amino acid residues of some ribosomal proteins and translation factors in both eukaryotes and eubacteria have accumulated. The reaction is catalyzed by dedicated oxygenases (so-called ribosomal oxygenases), whose action is impaired under hypoxia conditions. The modification occurs at amino acid residues directly involved in the formation of the main functional sites of ribosomes and factors. This review summarizes currently available data on the specific hydroxylation of protein constituents of eukaryotic and eubacterial translation systems with a special emphasis on the human system, as well as on the links between hypoxia impacts on the operation of ribosomal oxygenases, the functioning of the translational apparatus and human health problems.

The small subunit ribosomal protein uS3 is a critically important player in the ribosome-mRNA interactions during translation and has numerous functions not directly related to protein synthesis in eukaryotes. A peculiar feature of the human uS3 protein is the ability of its fragment 55–64 exposed on the 40S subunit surface near the mRNA entry channel to form cross-links with 3′-terminal dialdehyde derivatives of various unstructured RNAs and with abasic sites in single-stranded DNAs. Here we showed that the ability of the above uS3 fragment to cross-link to abasic sites in DNAs is inherent only in mature cytoplasmic 40S subunits, but not nuclear pre-40S particles, which implies that it may be relevant to the ribosome-mRNA interplay. To clarify this issue, we investigated interactions of human ribosomes with synthetic mRNA analogues bearing an abasic site protected by a photocleavable group at the 3′-termini. We found that these mRNA analogues can form specific complexes with 80S ribosomes and 40S subunits, where the undamaged upstream part of the analogue is fixed in the mRNA binding channel by interaction with the P-site tRNA, and the downstream part located outside the ribosome is cross-linked to the uS3 fragment 55–64. The yield of cross-links of the mRNA analogues was rather high when their undamaged parts were bound to the mRNA channel prior to deprotection of the abasic site enabling its covalent attachment to the 40S subunit via the uS3 protein, but not vice versa. Based on our findings, one can assume that abasic sites, which can occur in mRNAs due to oxidative stress and ageing, are able to interact directly with the uS3 fragment exposed on the 40S subunit surface near the mRNA entry channel during translation. Consequently, the 40S subunit can be considered as a potential mRNA quality controller.

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.

The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 50-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position-3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position-3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes. (c) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.