Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site.

The internal ribosome entry site (IRES) RNA of bovine viral diarrhoea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modelling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA in solution forms a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are represented in tertiary interactions to form an H-type pseudoknot. Computational modelling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

New Insights on the Mobility of Viral and Host Non-Coding RNAs Reveal Extracellular Vesicles as Intriguing Candidate Antiviral Targets.

Intercellular communication occurring by cell-to-cell contacts and via secreted messengers trafficked through extracellular vehicles is critical for regulating biological functions of multicellular organisms. Recent research has revealed that non-coding RNAs can be found in extracellular vesicles consistent with a functional importance of these molecular vehicles in virus propagation and suggesting that these essential membrane-bound bodies can be highjacked by viruses to promote disease pathogenesis. Newly emerging evidence that coronaviruses generate non-coding RNAs and use extracellular vesicles to facilitate viral pathogenicity may have important implications for the development of effective strategies to combat COVID-19, a disease caused by infection with the novel coronavirus, SARS-CoV-2. This article provides a short overview of our current understanding of the interactions between non-coding RNAs and extracellular vesicles and highlights recent research which supports these interactions as potential therapeutic targets in the development of novel antiviral therapies.

Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis.

BACKGROUND: Trans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes. RESULTS: Complexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity. CONCLUSION: Our observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.

Comparative structural studies of bovine viral diarrhea virus IRES RNA.

The internal ribosomal entry site (IRES) RNA of bovine viral diarrhea virus (BVDV) has been implicated in virus propagation. To gain insight into the structure and potential function of the BVDV IRES RNA, we collected and aligned 663 of its sequences. Compensatory Watson-Crick and wobble G·U pairs were investigated to establish phylogenetically supported secondary structures for each of the BVDV IRES RNA sequences. The extensively folded BVDV IRES RNAs were composed of helices 2, 3 and 4. Helix 2 consisted of five helical sections. Helix 3 contained sections 3a to 3j as well as six helical insertions 3.1-3.6. Sections 3a and 3b together with helices 3.6 and 4 formed an RNA pseudoknot. Two highly variable regions corresponded to hairpins 3j and 3.4. Three-dimensional modeling of the BVDV-1b strain Osloss IRES RNA predicted an elongated structure with approximate dimensions of 170 Å by 65 Å by 90 Å. The model of the IRES RNA-ribosome complex suggested proximity between helix 2 of the BVDV IRES and ribosomal proteins S5 and S25.

In silico analysis of IRES RNAs of foot-and-mouth disease virus and related picornaviruses.

Foot-and-mouth disease virus (FMDV) uses an internal ribosome entry site (IRES), a highly structured segment of its genomic RNA, to hijack the translational apparatus of an infected host. Computational analysis of 162 type II picornavirus IRES RNA sequences yielded secondary structures that included only base pairs supported by comparative or experimental evidence. The deduced helical sections provided the foundation for a hypothetical three-dimensional model of FMDV IRES RNA. The model was further constrained by incorporation of data derived from chemical modification and enzymatic probing of IRES RNAs as well as high-resolution information about IRES RNA-bound proteins.

Visualizing the transfer-messenger RNA as the ribosome resumes translation.

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro.

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3' end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA- and mRNA-like domains that are usually connected by four pseudoknots (pk1-pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Deltah4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.

Making the jump: new insights into the mechanism of trans-translation.

The transfer-messenger ribonucleoprotein (tmRNP), which is composed of RNA and a small protein, small protein B (SmpB), recycles ribosomes that are stalled on broken mRNAs lacking stop codons and tags the partially translated proteins for degradation. Although it is not yet understood how the ribosome gets from the 3' end of the truncated message onto the messenger portion of the tmRNA to add the tag, a recent study in BMC Biology has shed some light on this astonishing feat.

The 5e motif of eukaryotic signal recognition particle RNA contains a conserved adenosine for the binding of SRP72.

The signal recognition particle (SRP) plays a pivotal role in transporting proteins to cell membranes. In higher eukaryotes, SRP consists of an RNA molecule and six proteins. The largest of the SRP proteins, SRP72, was found previously to bind to the SRP RNA. A fragment of human SRP72 (72c') bound effectively to human SRP RNA but only weakly to the similar SRP RNA of the archaeon Methanococcus jannaschii. Chimeras between the human and M. jannaschii SRP RNAs were constructed and used as substrates for 72c'. SRP RNA helical section 5e contained the 72c' binding site. Systematic alteration within 5e revealed that the A240G and A240C changes dramatically reduced the binding of 72c'. Human SRP RNA with a single A240G change was unable to form a complex with full-length human SRP72. Two small RNA fragments, one composed of helical section 5ef, the other of section 5e, competed equally well for the binding of 72c', demonstrating that no other regions of the SRPR RNA were required. The biochemical data completely agreed with the nucleotide conservation pattern observed across the phylogenetic spectrum. Thus, most eukaryotic SRP RNAs are likely to require for function an adenosine within their 5e motifs. The human 5ef RNA was remarkably resistant to ribonucleolytic attack suggesting that the 240-AUC-242 "loop" and its surrounding nucleotides form a peculiar compact structure recognized only by SRP72.

The tmRDB and SRPDB resources.

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Royal Veterinary and Agricultural University, Denmark (http://tmrdb.kvl.dk/). The signal recognition particle database (SRPDB) at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html is mirrored at http://srpdb.kvl.dk/ and the University of Goteborg (http://bio.lundberg.gu.se/dbs/SRPDB/SRPDB.html). The databases assist in investigations of the tmRNP (a ribonucleoprotein complex which liberates stalled bacterial ribosomes) and the SRP (a particle which recognizes signal sequences and directs secretory proteins to cell membranes). The curated tmRNA and SRP RNA alignments consider base pairs supported by comparative sequence analysis. Also shown are alignments of the tmRNA-associated proteins SmpB, ribosomal protein S1, alanyl-tRNA synthetase and Elongation Factor Tu, as well as the SRP proteins SRP9, SRP14, SRP19, SRP21, SRP54 (Ffh), SRP68, SRP72, cpSRP43, Flhf, SRP receptor (alpha) and SRP receptor (beta). All alignments can be easily examined using a new exploratory browser. The databases provide links to high-resolution structures and serve as depositories for structures obtained by molecular modeling.

Comparative 3-D modeling of tmRNA.

BACKGROUND: Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA. RESULTS: To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA. CONCLUSION: Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.

Transfer-messenger RNA unfolds as it transits the ribosome.

In bacteria, translation of mRNAs lacking stop codons produces truncated polypeptides and traps ribosomes in unproductive complexes. Potentially harmful truncated proteins are tagged with short peptides encoded by the mRNA-like domain of tmRNA and targeted for digestion by housekeeping proteases. We show that altered Escherichia coli transfer-messenger RNAs (tmRNAs) produce in vivo fusion proteins with peptide tags that extend far beyond the conventional termination signal of the wild-type tmRNA. Regions of tmRNA capable of serving as templates for protein synthesis include helix 5, as well as pseudoknots 2, 3, and 4. The removal of all six in-frame stop codons negatively affects tmRNA processing, thereby preventing translation of the 3' portion of the tRNA-like domain. These findings provide evidence that trans-translation can be accompanied by the unfolding of significant portions of the tmRNA molecule. Many of these conformational changes are likely to be required during trans-translation to maintain the ribosomal subunits in close proximity to the tmRNA for monitoring its transit.

Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA.

Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds). In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes. The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA. Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity. Furthermore, the E. coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E. coli S1, showed no interaction with E. coli tmRNA, as judged by the results of a gel shift assay. Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)). These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.

Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation.

Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA- and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in trans-translation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.

SmpB: a protein that binds to double-stranded segments in tmRNA and tRNA.

Binding of the SmpB protein to tmRNA is essential for trans-translation, a process that facilitates peptide tagging of incompletely synthesized proteins. We have used three experimental approaches to study these interactions in vitro. Gel mobility shift assays demonstrated that tmRNA(Delta90-299), a truncated tmRNA derivative lacking pseudoknots 2-4, has the same affinity for the Escherichia coli and Aquifex aeolicus SmpB proteins as the intact E. coli tmRNA. These interactions can be challenged by double-stranded RNAs such as tRNAs and 5S rRNA and are abolished by removal of 24 amino acids from the C-terminus of the A. aeolicus protein. A combination of enzymatic probing and UV-induced cross-linking showed that three SmpB molecules can bind to a single tmRNA(Delta90-299) and tRNA molecule. Irradiation of E. coli tmRNA and yeast tRNA(Phe) bound to a single SmpB molecule with UV light induced cross-links to residues C343 and m(1)A48, respectively, in their T-loops and to their 3' terminal adenosines. These findings indicate that the acceptor-T arm constitutes the primary SmpB binding site in both tmRNA and tRNA. The remaining two SmpB molecules associate with the anticodon stem-like region of tmRNA and the anticodon arm of tRNAs. As the T and anticodon loops are dispensable for SmpB binding, it seems that SmpB recognizes double helical segments in both tmRNA and tRNA molecules. Although these interactions involve analogous elements in both molecules, their different effects on aminoacylation appear to reflect subtle structural differences between the tRNA-like domain of tmRNA and tRNA.