Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are ...currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.
•Cytosolic proteins YB-1 and NSUN2 recognize specific motifs found in exosomal RNAs.•Proteins YB-1 and NSUN2 are present in exosomes secreted by HEK293 cells.•mRNA species containing motifs recognized by YB-1 and NSUN2 are enriched in exosomes.
A number of mutations in the
gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected ...HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g.,
and
. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.
The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting ...its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.
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 eS26 motif YxxPKxYxK controls the content of light polysomes in mammalian cells.•The eS26 motif YxxPKxYxK provides the binding of eRF3e to 80S elongating ribosomes.•The eS26 motif YxxPKxYxK role is related to eIF3e-promoted selective translation.
Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA ...interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3' untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.
Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, ...using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES-rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.
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.
•Damaged mRNA cross-links to ribosomal protein uS3 via abasic site during translation.•mRNA abasic site cross-links to uS3 when arrives to the certain favorable position.•The 55–64 peptide of uS3 inspects mRNAs for the presence of abasic sites during translation.•Abasic site-uS3 cross-linking is one of ways of the ribosome-based mRNA surveillance.
Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3′-terminus to investigate mutual arrangement of the main components of ...translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3′-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.
•eRF3 remains bound at the ribosome after GTP hydrolysis and peptide release.•CCA-end of P/P tRNA contacts eRF1 M domain similarly at all post-termination steps.•eRF1 N domain is similarly arranged toward the stop codon at all termination steps.
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.
•Rp uS3 cross-links to DNA abasic site in mature 40S but not in pre-40S particles.•Abasic site in mRNA can cross-link to 40S subunits via uS3.•Abasic site in mRNA interacts with an exposed uS3 peptide near the mRNA entry site.•Ribosomal complex with mRNA cross-linked to uS3 is a potential target for no-go decay.
Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires ...site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron-electron resonance provided spin-spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.
PDF
