Deficiency of the ribosomal protein uS10 (RPS20) reorganizes human cells translatome according to the abundance, CDS length and GC content of mRNAs.

Ribosomal protein uS10, a product of the RPS20 gene, is an essential constituent of the small (40S) subunit of the human ribosome. Disruptive mutations in its gene are associated with a predisposition to hereditary colorectal carcinoma. Here, using HEK293T cells, we show that a deficiency of this protein leads to a decrease in the level of ribosomes (ribosomal shortage). RNA sequencing of the total and polysome-associated mRNA samples reveals hundreds of genes differentially expressed in the transcriptome (t)DEGs and translatome (p)DEGs under conditions of uS10 deficiency. We demonstrate that the (t)DEG and (p)DEG sets partially overlap, determine genes with altered translational efficiency (TE) and identify cellular processes affected by uS10 deficiency-induced ribosomal shortage. We reveal that translated mRNAs of upregulated (p)DEGs and genes with altered TE in uS10-deficient cells are generally more abundant and that their GC contents are significantly lower than those of the respective downregulated sets. We also observed that upregulated (p)DEGs have longer coding sequences. Based on our findings, we propose a combinatorial model describing the process of reorganization of mRNA translation under conditions of ribosomal shortage. Our results reveal rules according to which ribosomal shortage reorganizes the transcriptome and translatome repertoires of actively proliferating cells.

Functional involvement of a conserved motif in the middle region of the human ribosomal protein eL42 in translation.

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.

Mutation at the Site of Hydroxylation in the Ribosomal Protein uL15 (RPL27a) Causes Specific Changes in the Repertoire of mRNAs Translated in Mammalian Cells.

Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation. By applying RNA-seq to the total cellular and polysome-associated mRNAs, we identified differentially expressed genes (DEGs) in cells containing exogenous uL15 or its mutant form. Analyzing mRNA features of up- and down-regulated DEGs, we found an increase in the level of more abundant mRNAs and shorter CDSs in cells with uL15 mutant for both translated and total cellular mRNAs. The level of longer and rarer mRNAs, on the contrary, decreased. Our data show how ribosome heterogeneity can change the composition of the translatome and transcriptome, depending on the properties of the translated mRNAs.

Many Faces of Next-Generation Sequencing in Gene Expression Studies.

Gene expression is a fundamental cellular process that ensures the transfer of information encoded in a gene into the final functional product [...].

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs.

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 functional role of the eukaryote-specific motif YxxPKxYxK of the human ribosomal protein eS26 in translation.

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.

Changes in the Transcriptome Caused by Mutations in the Ribosomal Protein uS10 Associated with a Predisposition to Colorectal Cancer.

A number of mutations in the RPS20 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., PPM1D and PIGN. 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.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells.

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.

Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy.

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.

Knockdown of the Ribosomal Protein eL38 in HEK293 Cells Changes the Translational Efficiency of Specific Genes.

The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals.

Knockdown of the mRNA encoding the ribosomal protein eL38 in mammalian cells causes a substantial reorganization of genomic transcription.

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription. An approximately 4-fold decrease in the level of eL38 was shown to cause changes in the expression of nearly 1500 genes. Among the down-regulated genes, there were those responsible for p53 activity, Ca2+ metabolism and several signaling processes, as well as genes involved in the organization and functioning of the cytoskeleton. The genes related to rRNA processing and translation, along with many others, including those whose dysregulation is associated with developmental disorders, turned out to be up-regulated. Thus, we demonstrated that the decreased RPL38 expression leads to a significant reorganization of genomic transcription. Our findings suggest a possible link between the balance of eL38 and genes implicated in osteogenesis, thereby contributing to the elucidation of the reasons for the appearance of the above Ts mutant phenotype in animals.

The human ribosomal protein eL29 binds in vivo to the cognate mRNA by interacting with its coding sequence, as revealed from in-cell cross-linking data.

The balance of ribosomal proteins is important for the assembly of ribosomal subunits and cell viability. The synthesis of ribosomal proteins in a eukaryotic cell is controlled by various mechanisms, including autoregulation, which so far has been revealed for only a few of these proteins. We applied the photoactivatable 4-thiouridine-enhanced cross-linking and immunoprecipitation assay to HEK293T cells overproducing FLAG-labeled human ribosomal protein eL29 (eL29FLAG) to determine which RNAs other than rRNA interact with eL29. We demonstrated that eL29FLAG was incorporated into 60S subunits, and that ribosomes with those containing eL29FLAG were competent in translation. Analysis of the next generation sequencing data obtained from a DNA library derived from RNA fragments with covalently attached eL29FLAG peptide residues showed that the protein was cross-linked to the mRNA of the eL29-coding gene, which turned out to be its only major RNA target. The eL29FLAG cross-linking sites were located in the 3' part of the mRNA coding sequence (CDS). A specific helix that mimics the eL29 binding site on 28S rRNA was proposed as a site that is recognized by the protein upon its binding to the cognate mRNA. In addition, it was found that both eL29FLAG mRNA and eL29 mRNA, unlike those of other ribosomal proteins, were co-immunoprecipitated with eL29FLAG from the ribosome-depleted cell lysate, and recombinant eL29 inhibited the translation of the eL29 mRNA CDS transcript in a cell-free system. All this suggests that human eL29 regulates its own synthesis via a feedback mechanism by binding to the cognate mRNA, preventing its translation.

Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level.

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Degenerate consensus sequences in the 3'-untranslated regions of cellular mRNAs as specific motifs potentially involved in the YB-1-mediated packaging of these mRNAs.

The multifunctional protein YB-1 has previously been shown to be the only protein of the cytoplasmic extract of HEK293 cells, which is able to specifically interact with imperfect RNA hairpins containing motifs that are often found in exosomal (e) RNAs. In addition, it has been revealed that similar hairpins formed by degenerate consensus sequences corresponding to three eRNA-specific motifs are responsible for the cooperative binding of YB-1 to RNA in vitro. Here, using the photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation method applied to HEK293 cells producing FLAG-labeled YB-1, we identified mRNAs cross-linked to YB-1 in vivo and then carried out a search for the aforementioned sequences in the regions of the YB-1 cross-linking sites. It turned out that many of the mRNAs found cross-linked to YB-1 encode proteins associated with various regulatory processes, including responses to stress. More than half of all cross-linked mRNAs contained degenerate consensus sequences, which were preferably located in 3'-untranslated regions (UTRs), where most of the YB-1 cross-linking sites appeared, although not close to these sequences. Furthermore, YB-1 was mainly cross-linked to those mRNAs with degenerate consensus sequences, which could be classified as packaged because their translation levels were low compared to cellular levels. This suggests that the cooperative binding of YB-1 to mRNAs through the above sequences probably triggers the well-known multimerization of YB-l, leading to the packaging of these mRNAs. Thus, our findings indicate a previously unknown link between the degenerate consensus sequences present in the 3'-UTRs of many cytoplasmic mRNAs and YB-1-mediated translational silencing.

mRNA regions where 80S ribosomes pause during translation elongation in vivo interact with protein uS19, a component of the decoding site.

In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.

Exploring the interactions of short RNAs with the human 40S ribosomal subunit near the mRNA entry site by EPR spectroscopy.

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.

Arrangements of nucleotides flanking the start codon in the IRES of the hepatitis C virus in the IRES binary complex with the human 40S ribosomal subunit.

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 5'-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.

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.

Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy.

The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.

Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting.

We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1-3 in the HEK293 cell mRNA 3' UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3' UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.