The global and local distribution of RNA structure throughout the SARS-CoV-2 genome

Abstract

<jats:title>Abstract</jats:title><jats:p>SARS-CoV-2 is the causative viral agent of COVID-19, the disease at the center of the current global pandemic. While knowledge of highly structured regions is integral for mechanistic insights into the viral infection cycle, very little is known about the location and folding stability of functional elements within the massive, ~30kb SARS-CoV-2 RNA genome. In this study, we analyze the folding stability of this RNA genome relative to the structural landscape of other well-known viral RNAs. We present an in-silico pipeline to locate regions of high base pair content across this long genome and also identify well-defined RNA structures, a method that allows for direct comparisons of RNA structural complexity within the several domains in SARS-CoV-2 genome. We report that the SARS-CoV-2 genomic propensity to stable RNA folding is exceptional among RNA viruses, superseding even that of HCV, one of the most highly structured viral RNAs in nature. Furthermore, our analysis reveals varying levels of RNA structure across genomic functional regions, with accessory and structural ORFs containing the highest structural density in the viral genome. Finally, we take a step further to examine how individual RNA structures formed by these ORFs are affected by the differences in genomic and subgenomic contexts. The conclusions reported in this study provide a foundation for structure-function hypotheses in SARS-CoV-2 biology, and in turn, may guide the 3D structural characterization of potential RNA drug targets for COVID-19 therapeutics.</jats:p>

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

“…One can make two major observations about the global architecture the SARS-CoV-2 genome. First, this in-vivo derived, SHAPE-constrained model strongly agrees with the high doublestrand RNA content predicted from the entirely in silico model recently reported by our lab (Tavares et al, 2020). Because the data herein were obtained in-vivo, this work confirms that the unusually high double-strand content is maintained in a cellular context.…”

“…One important cautionary observation from our work is the poor correlation of SHAPE reactivities between two in-vivo biological replicates for regions encoding the subgenomic RNAs. Previous in silico work from our lab has shown that individual subgenomic RNAs (sgRNAs), such as the N sgRNA, fold differently than the corresponding regions in the genomic RNA due to differences in upstream sequence context( Tavares et al, 2020 ). Though our tiled-amplicon design affords sequencing coverage for the entire SARS-CoV-2 genome, it precludes deconvolution of reactivity signals for regions shared between genomic- and subgenomic RNAs.…”

“…Across the sub genomic RNA ORFs, the Pearson 's correlation is poor. We believe this reflects the fact that Amplicons 13, 14, 15, and 16 will amplify both full-length and sub-genomic RNAs, and the difference in context will result in different secondary structures (Tavares et al, 2020). For this reason, despite the fact all data have been obtained globally, subsequent discrete structural analysis will focus on shared features of the viral termini and the Orf1ab region.…”

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

“…Besides the well-characterized structures within the 5′ and 3′ UTR, and the FSE, the SARS-CoV-2 genome is predicted to have an exceptionally high propensity to form stable RNA structures, significantly higher compared with that of other RNA viruses, including Hepatitis C virus (HCV), one of the most highly structured viral RNAs to date [ 72 ]. Indeed, recent genome-scale structure analyses of the SARS-CoV-2 genome, both in vitro and in living infected host cells, have led to the identification of a plethora of novel highly stable RNA structure elements, spread along the whole genome.…”

The architecture of the SARS-CoV-2 RNA genome inside virion

“…We rst determined to validate the viral RNA spatial interactions revealed by vRIC-seq. For this purpose, we deduced several conserved RNA duplexes in coronavirus using vRIC-seq data and compared it with recently proposed SARS-CoV-2 secondary structure models 13,[21][22][23][24][34][35][36] . As expected, vRIC-seq faithfully recapitulated all of the stem-loops in the 5' UTR (1-395 nt) of SARS-CoV-2 except stem-loop 1 (SL1), which is approximately 40 nt in length and can't be puri ed by AMPure XP beads (Fig.…”