Structural 3D Domain Reconstruction of the RNA Genome from Viruses with Secondary Structure Models

Three-dimensional RNA domain reconstruction is important for the assembly, disassembly and delivery functionalities of a packed proteinaceus capsid. However, to date, the self-association of RNA molecules is still an open problem. Recent chemical probing reports provide, with high reliability, the secondary structure of diverse RNA ensembles, such as those of viral genomes. Here, we present a method for reconstructing the complete 3D structure of RNA genomes, which combines a coarse-grained model with a subdomain composition scheme to obtain the entire genome inside proteinaceus capsids based on secondary structures from experimental techniques. Despite the amount of sampling involved in the folded and also unfolded RNA molecules, advanced microscope techniques can provide points of anchoring, which enhance our model to include interactions between capsid pentamers and RNA subdomains. To test our method, we tackle the satellite tobacco mosaic virus (STMV) genome, which has been widely studied by both experimental and computational communities. We provide not only a methodology to structurally analyze the tertiary conformations of the RNA genome inside capsids, but a flexible platform that allows the easy implementation of features/descriptors coming from both theoretical and experimental approaches.

The mitochondrial genome of the semi-slug Omalonyx unguis (Gastropoda: Succineidae) and the phylogenetic relationships within Stylommatophora

Here we report the first complete mitochondrial genome of the semi-slug Omalonyx unguis (d’Orbigny, 1836) (Gastropoda: Succineidae). Sequencing was performed on a specimen from Argentina. Assembly was performed using Sanger data and Illumina next generation sequencing (NGS). The mitogenome was 13,984 bp in length and encoded the 37 typical Metazoan genes. A potential origin for mitochondrial DNA replication was found in a non-coding intergenic spacer (49 bp) located between cox3 and tRNA-Ile genes, and its secondary structure was characterized. Secondary structure models of the tRNA genes of O. unguis largely agreed with those proposed for other mollusks. Secondary structure models for the two rRNA genes were also obtained. To our knowledge, the 12S-rRNA model derived here is the first complete one available for mollusks. Phylogenetic analyses based on the mitogenomes of O. unguis and 37 other species of Stylommatophora were performed using amino acid sequences from the 13 protein-coding genes. Our results located Succineoidea as a sister group of Helicoidea + Urocoptoidea, similar to previous studies based on mitochondrial genomes. The gene arrangement of O. unguis was identical to that reported for another species of Succineoidea. The unique rearrangements observed for this group within Stylommatophora, may constitute synapomorphies for the superfamily.

Relative Information Gain: Shannon entropy-based measure of the relative structural conservation in RNA alignments

Structural characterization of RNAs is a dynamic field, offering many modelling possibilities. RNA secondary structure models are usually characterized by an encoding that depicts structural information of the molecule through string representations or graphs. In this work, we provide a generalization of the BEAR encoding (a context-aware structural encoding we previously developed) by expanding the set of alignments used for the construction of substitution matrices and then applying it to secondary structure encodings ranging from fine-grained to more coarse-grained representations. We also introduce a re-interpretation of the Shannon Information applied on RNA alignments, proposing a new scoring metric, the Relative Information Gain (RIG). The RIG score is available for any position in an alignment, showing how different levels of detail encoded in the RNA representation can contribute differently to convey structural information. The approaches presented in this study can be used alongside state-of-the-art tools to synergistically gain insights into the structural elements that RNAs and RNA families are composed of. This additional information could potentially contribute to their improvement or increase the degree of confidence in the secondary structure of families and any set of aligned RNAs.

IPANEMAP: integrative probing analysis of nucleic acids empowered by multiple accessibility profiles

The manual production of reliable RNA structure models from chemical probing experiments benefits from the integration of information derived from multiple protocols and reagents. However, the interpretation of multiple probing profiles remains a complex task, hindering the quality and reproducibility of modeling efforts. We introduce IPANEMAP, the first automated method for the modeling of RNA structure from multiple probing reactivity profiles. Input profiles can result from experiments based on diverse protocols, reagents, or collection of variants, and are jointly analyzed to predict the dominant conformations of an RNA. IPANEMAP combines sampling, clustering and multi-optimization, to produce secondary structure models that are both stable and well-supported by experimental evidences. The analysis of multiple reactivity profiles, both publicly available and produced in our study, demonstrates the good performances of IPANEMAP, even in a mono probing setting. It confirms the potential of integrating multiple sources of probing data, informing the design of informative probing assays.

IPANEMAP: Integrative Probing Analysis of Nucleic Acids Empowered by Multiple Accessibility Profiles

The manual production of reliable RNA structure models from chemical probing experiments benefits from the integration of information derived from multiple protocols and reagents. However, the interpretation of multiple probing profiles remains a complex task, hindering the quality and reproducibility of modeling efforts. We introduce IPANEMAP, the first automated method for the modeling of RNA structure from multiple probing reactivity profiles. Input profiles can result from experiments based on diverse protocols, reagents, or collection of variants, and are jointly analyzed to predict the dominant conformations of an RNA. IPANEMAP combines sampling, clustering, and multi-optimization, to produce secondary structure models that are both stable and well-supported by experimental evidences. The analysis of multiple reactivity profiles, both publicly available and produced in our study, demonstrates the good performances of IPANEMAP, even in a mono probing setting. It confirms the potential of integrating multiple sources of probing data, informing the design of informative probing assays. Availability: IPANEMAP is freely downloadable at https://github.com/afafbioinfo/IPANEMAP Contact: [email protected]

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 ‘SARS-related-conserved’ regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 ‘SARS-CoV-2-conserved-structured’ regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5’ UTR, frame-shifting element, and 3’ UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 ‘SARS-CoV-2-conserved-unstructured’ genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

The complete mitochondrial genome of Amphinemura claviloba (Wu, 1973) (Plecoptera: Nemouridae)

We sequenced the complete mitochondrial genome (mitogenome) of a stonefly, Amphinemura claviloba (Wu, 1973), of the family Nemouridae (Insecta: Plecoptera). The mitogenome was 15,707 bp long and contained typical 37 genes with an A+T content of 68.5%. All protein-coding genes (PCGs) use standard initiation codons (methionine and isoleucine), except ND1 and ND5 which starts with TTG and GTG, respectively. Two of the 13 PCGs harbor the incomplete termination codon. All tRNA genes have typical clover secondary structures, except the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. Secondary structure models of the ribosomal RNA genes of A. claviloba are similar to those proposed for other insects. We also found some structural elements in the control region, such as tandem repeats, poly-C stretch and microsatellite-like elements, etc. Phylogenetic analyses showed the clades for the Nemoura, Amphinemura, and (Mesonemoura + Sphaeronemoura + Indonemoura + Protonemura) are well supported in a polytomy.

Combining phase information in reciprocal space for molecular replacement with partial models

ARCIMBOLDOallowsab initiophasing of macromolecular structures below atomic resolution by exploiting the location of small model fragments combined with density modification in a multisolution frame. The model fragments can be either secondary-structure elements predicted from the sequence or tertiary-structure fragments. The latter can be derived from libraries of typical local folds or from related structures, such as a low-homology model that is unsuccessful in molecular replacement. In allARCIMBOLDOapplications, fragments are searched for sequentially. Correct partial solutions obtained after each fragment-search stage but lacking the necessary phasing power can, if combined, succeed. Here, an analysis is presented of the clustering of partial solutions in reciprocal space and of its application to a set of different cases. In practice, the task of combining model fragments from anARCIMBOLDOrun requires their referral to a common origin and is complicated by the presence of correct and incorrect solutions as well as by their not being independent. TheF-weighted mean phase difference has been used as a figure of merit. Clustering perfect, non-overlapping fragments dismembered from test structures in polar and nonpolar space groups shows that density modification before determining the relative origin shift enhances its discrimination. In the case of nonpolar space groups, clustering ofARCIMBOLDOsolutions from secondary-structure models is feasible. The use of partially overlapping search fragments provides a more favourable circumstance and was assessed on a test case. Applying the devised strategy, a previously unknown structure was solved from clustered correct partial solutions.