In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the ...translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel. Chemical probing with BzCN revealed that h16 in the 48S PICs containing eIF3j or scanning factor DHX29 is strongly shielded; the effect was observed with all the mRNAs used, and h16 remained protected as well in 80S post-initiation complexes lacking these factors. Altogether, the obtained results allowed us to suggest that eIF3j bound at the 48S PICs makes the rp uS3 inaccessible for binding of RNAs and this factor subunit is responsible for the decrease of h16 conformational flexibility; the latter is manifested as reduced accessibility of h16 to BzCN. Thus, our findings provide new insights into how eIF3j is implicated in ensuring the proper conformation of the mRNA entry channel, thereby facilitating mRNA loading.
•RNA-binding fragment of rp uS3 is shielded by eIF3j in 48S preinitiation complexes.•Ribosomal eIF3j and mRNA sites overlap since third triplet of mRNA coding sequence.•Binding of eIF3j and DHX29 to 40S subunits promotes rigid state of 18S rRNA helix 16.
Abstract
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.
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.
•In the 40S·HCV IRES complex, the IRES base at position −3 adjoins uS11 and eS26•The HCV IRES base at positions +4/5 hardly comes close to uS19, an A site element.•The HCV IRES coding part is correctly arranged in a tiny fraction of binary complexes.
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.
•The main target RNA of the human ribosomal protein eL29 in cells is its own mRNA.•eL29 interacts with the 3′-terminal region of the eL29 mRNA coding sequence.•Excess eL29 binds to its own mRNA when it is not translated by ribosomes.•Recombinant eL29 inhibits the in vitro translation of eL29 mRNA transcript.•A putative binding site of eL29 in the cognate mRNA mimics its site on 28S rRNA.
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.
Evolutionary potential of viruses can result in outbreaks of well‐known viruses and emergence of novel ones. Pharmacological methods of intervening the reproduction of various less popular, but not ...less important viruses are not available, as well as the spectrum of antiviral activity for most known compounds. In the framework of chemical biology paradigm, characterization of antiviral activity spectrum of new compounds allows to extend the antiviral chemical space and provides new important structure–activity relationships for data‐driven drug discovery. Here we present a primary assessment of antiviral activity of spiro‐annulated derivatives of seven‐membered heterocycles, oxepane and azepane, in phenotypic assays against viruses with different genomes, virion structures, and genome realization schemes: orthoflavivirus (tick‐borne encephalitis virus, TBEV), enteroviruses (poliovirus, enterovirus A71, echovirus 30), adenovirus (human adenovirus C5), hantavirus (Puumala virus). Hit compounds inhibited reproduction of adenovirus C5, the only DNA virus in the studied set, in the yield reduction assay, and did not inhibit reproduction of RNA viruses.
Diversity‐oriented antiviral activity screening revealed adenovirus yield reduction by a structurally novel chemotype of spiro‐annulated oxepanes and azepenes.
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.
Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far ...only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3′-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA–protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed.
•Binding of human 40S ribosome proteins to the major 3′ domain of 18S rRNA was studied.•In vitro reconstitution of the 40S ribosomal subunit head was performed.•Role of the unstructured extensions of r-proteins in RNP aggregation is discussed.
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•DEER reveals the conformational variability of mRNA at the certain translation steps.•Elongation and termination complexes exist in 2 conformations in dynamic equilibrium.•The ...conformations of mRNA in 40S channel undergo no major change during termination.
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.
To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The ...human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.
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