Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org.

Rfam 14: expanded coverage of metagenomic, viral and microRNA families.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

/pdf/direct-rna-nanopore-sequencing-of-full-length-coronavirus-4za86m4mz0.pdf

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5' UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

/pdf/genome-wide-mapping-of-sars-cov-2-rna-structures-identifies-4z5o3tufgm.pdf

Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements.

Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

/pdf/secondary-structure-determination-of-conserved-sars-cov-2-4urr4r12q2.pdf

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions.

ABSTRACT In Sinorhizobium meliloti, the methionine biosynthesis genes metA and metZ are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the metZ riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the metA riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the metA riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to metA regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the metA and metZ promoters and the steady-state levels of the read-through metA and metZ mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine–Dalgarno (SD) sequence in the metA riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate metA translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal metA and metZ mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.

Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance

Abstract The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

/pdf/unraveling-the-small-proteome-of-the-plant-symbiont-39qcbz6d.pdf

Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics

Many Gram-negative pathogens utilize the type III secretion system (T3SS) to translocate virulence-promoting effector proteins into eukaryotic host cells. The activity of this system results in a severe reduction of bacterial growth and division, summarized as secretion-associated growth inhibition (SAGI). In Yersinia enterocolitica, the T3SS and related proteins are encoded on a virulence plasmid. We identified a ParDE-like toxin–antitoxin system on this virulence plasmid in genetic proximity to yopE, encoding a T3SS effector. Effectors are strongly upregulated upon activation of the T3SS, indicating a potential role of the ParDE system in the SAGI or maintenance of the virulence plasmid. Expression of the toxin ParE in trans resulted in reduced growth and elongated bacteria, highly reminiscent of the SAGI. Nevertheless, the activity of ParDE is not causal for the SAGI. T3SS activation did not influence ParDE activity; conversely, ParDE had no impact on T3SS assembly or activity itself. However, we found that ParDE ensures the presence of the T3SS across bacterial populations by reducing the loss of the virulence plasmid, especially under conditions relevant to infection. Despite this effect, a subset of bacteria lost the virulence plasmid and regained the ability to divide under secreting conditions, facilitating the possible emergence of T3SS-negative bacteria in late acute and persistent infections.

/pdf/a-parde-toxin-antitoxin-system-is-responsible-for-the-bo2ox27f.pdf

A ParDE toxin–antitoxin system is responsible for the maintenance of the Yersinia virulence plasmid but not for type III secretion-associated growth inhibition

The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated, and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, cataloging the full complement of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with high confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search databases (iPtgxDBs). Searches against a standard and a 20-fold smaller Ribo-seq data-informed custom iPtgxDB confirmed many annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by applying MS and Ribo-seq as complementary approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

Robina Scheuer

Papers

Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions.

Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance

Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics

A ParDE toxin–antitoxin system is responsible for the maintenance of the Yersinia virulence plasmid but not for type III secretion-associated growth inhibition

Small proteome of the nitrogen-fixing plant symbiont Sinorhizobium meliloti