Abstract
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.
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.
•Cytosolic YB-1 binds to a construct corresponding to the 3′-UTR of exosomal mRNA.•Exosomal RNA-specific motifs contribute to the cooperative binding of YB-1 to mRNA.•YB-1 recognizes consensus sequences of specific hairpins in mRNA 3′ UTRs.
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.
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.
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.
•The main cellular RNA partners of YB-1 are identified using the PAR CLIP method.•Many YB-1 mRNA partners are associated with genes involved in the stress response.•YB-1 cross-linking sites and specific consensus sequences are mainly in 3′-UTRs.•mRNAs containing consensus sequences cross-link to YB-1, mostly when packed.
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.
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.
•Reduced level of ribosomal protein eL38 in cells largely reorganizes transcription.•eL38 knockdown down-regulates genes responsible for p53 activity.•eL38 knockdown activates genes associated with rRNA processing and translation.•BMP2 and BMP6 genes involved in osteogenesis are activated at a reduced eL38 level.
mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with ...nitroxide spin labels attached to the 5′-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNAPhe, which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.
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.
•Nucleophosmin binds to 40S ribosomal subunits in the presence of a nuclear extract.•Nucleophosmin binds to ribosomal protein eS26 in the presence of a nuclear extract.•C-terminal FLAG-modified eS26 reduces the content of ribosome-bound nucleophosmin.
Protein uL2 is essential for the catalytic activity of the ribosome and has a conserved shape in ribosomes from all domains of life. However, the sequence of its unstructured C‐terminal loop apex ...that contacts the conserved 23S/28S rRNA helix (H) 93 near the ribosomal peptidyl transferase center differs in bacteria, archaea and eukaryotes. Eukaryote‐specific residue His216 located in this loop in mammalian uL2 is hydroxylated in ribosomes. We used a set of chemical probes to explore the structure of an RNA that mimicked a segment of 28S rRNA domain V containing part of the uL2 binding site including H93, complexed with either natural (hydroxylated) or recombinant (unmodified) human uL2. It was found that both protein forms engage H93 during binding, but only natural uL2 (uL2n) protects it from hydroxyl radicals. The association of uL2n with RNA leads to changes in its structure at U4532 adjacent to the universally conserved U4531 (U2585, Escherichia coli numbering) involved in peptidyl transferase center formation, and at the universally conserved C4447 (2501) located in the ribosome near A4397 (2451) and C3909 (2063) belonging to the peptidyl transferase center. As a result, both nucleotides become strongly exposed to hydroxyl radicals. Our data argue that the hydroxyl group at His216 in the C‐terminal loop apex of mammalian uL2 contributes to stabilization of a protein conformation that is favorable for binding to H93 of 28S rRNA and that this binding induces structural rearrangement in the regions close to the peptidyl transferase center in the mature ribosome.
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