Mutation hotspots of SARS-CoV-2 RNA motifs conserved in betacoronaviruses

The pandemic of the coronavirus infection COVID-19, which began at the end of 2019 and caused by the SARS-CoV-2 virus, has led to unprecedented consequences in the world. By the end of May 2021, in the world there were 167 million infected and 3.5 million died directly from infection [1]. SARS-CoV-2 is a beta coronavirus, so it shares many conserved fragments with other known viruses of this type [2]. Since the beginning of the spread of the COVID-19, one of the important issues of research of the SARS-CoV-2 virus has been the search for its conserved RNA motifs and their functional annotation. These motifs are potential targets for the treatment and diagnosis of a disease caused by the virus. This report examines the structural RNA fragments of SARS-CoV-2, similar to the corresponding fragments in other beta coronaviruses [2]. For these RNA motifs the nucleotide variability during the spread of the virus, depending on their secondary structure, was investigated. All the motifs display the similar background variability although contain hypervariable positions.

New Short RNA Motifs Potentially Relevant in the Severe Acute Respiratory Syndrome Coronavirus 2 Genome

As time passes, identifying new pharmacological targets is becoming more difficult. Shortly, it will be necessary to devise new strategies to tackle the problem. The coronavirus disease outbreak caused by the severe acute respiratory syndrome coronavirus 2 , represents a threat to human health serving as example from what we just said. The present study was aimed to collect a set of short RNA motifs with potential biological impact, most of which have not been observed heretofore. Categorizing RNA triplets by their gross-composition, the study collected 88 short RNA motifs, shared by most coronavirus genera independent on the percent identity between genomes. Selected motifs contain all nearest-neighbours of the triplets A, T, G and A, C, G. The high percent identity between severe acute respiratory syndrome coronavirus genomes makes it difficult these peptides to be found by current methods. The results provide 50 motifs in the 1a polyprotein-encoding orf, 27 in the 1b polyprotein-encoding orf, 5 in the spike-encoding orf and 6 in the nucleocapsid-encoding orf. They also provide insights about the validity of the procedure, confirming some motifs interspersed or attached to known relevant functional fragments of the genome, although most of them have not yet been associated to any known function. The high level of preservation of these motifs in most coronavirus genera suggest they might have potential to be used for diagnostic, in vaccines, or as substrate for protease inhibitors.

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.

High-throughput dissection of the thermodynamic and conformational properties of a ubiquitous class of RNA tertiary contact motifs

Despite RNA’s diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural “motifs”—helices, junctions, and tertiary contact modules—to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used “RNA on a massively parallel array” (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

Fuzzy RNA-recognition by the Trypanosoma brucei editosome

The recognition of RNA-molecules by proteins and protein complexes is a critical step on all levels of gene expression. Typically, the generated ribonucleoprotein complexes rely on the binary interaction of defined RNA-sequences or precisely folded RNA-motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA-molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome we identified multiple similar but non-identical RNA-motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.523 (λ2). We establish that a synthetic 4V(3E)-RNA is sufficient to compete for the editosomal pre-mRNA binding site and that it is able to inhibit RNA-editing in vitro. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA-folding space by recognizing a fuzzy continuum of RNA-folds that fit a consensus graph-descriptor. This provides a mechanism on how the protein complex is able to bind the structurally pleomorphic pool of pre- and partially edited mRNAs. We speculate that other fuzzy RNA-recognition motifs exist especially for proteins that interact with multiple RNA-species.

Diversity and prevalence of ANTAR RNAs across actinobacteria

Background Computational approaches are often used to predict regulatory RNAs in bacteria, but their success is limited to RNAs that are highly conserved across phyla, in sequence and structure. The ANTAR regulatory system consists of a family of RNAs (the ANTAR-target RNAs) that selectively recruit ANTAR proteins. This protein-RNA complex together regulates genes at the level of translation or transcriptional elongation. Despite the widespread distribution of ANTAR proteins in bacteria, their target RNAs haven’t been identified in certain bacterial phyla such as actinobacteria. Results Here, by using a computational search model that is tuned to actinobacterial genomes, we comprehensively identify ANTAR-target RNAs in actinobacteria. These RNA motifs lie in select transcripts, often overlapping with the ribosome binding site or start codon, to regulate translation. Transcripts harboring ANTAR-target RNAs majorly encode proteins involved in the transport and metabolism of cellular metabolites like sugars, amino acids and ions; or encode transcription factors that in turn regulate diverse genes. Conclusion In this report, we substantially diversify and expand the family of ANTAR RNAs across bacteria. These findings now provide a starting point to investigate the actinobacterial processes that are regulated by ANTAR.

RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.