Computational Analysis of Conserved RNA Secondary Structure in Transcriptomes and Genomes

Abstract

Transcriptomics experiments and computational predictions both enable systematic discovery of new functional RNAs, but many putative noncoding transcripts arise instead from artifacts and biological noise, and current computational prediction methods have high false positive rates. I discuss prospects for improving computational methods for analyzing and identifying functional RNAs, with a focus on detecting signatures of conserved RNA secondary structure. An interesting new front is the application of chemical and enzymatic RNA structure probing experiments on a transcriptome-wide scale. I review several proposed approaches for incorporating structure probing data into computational RNA secondary structure prediction. Using probabilistic inference formalisms, I show how all these approaches can be unified in a well-principled framework. Using that framework, RNA probing data can easily be integrated into a wide range of different analyses that depend on RNA secondary structure inference, including homology search and genome-wide detection of new structural RNAs.

RNA structure inference through chemical mapping after accidental or intentional mutations

“…Despite these advances, the accuracy of RNA structure inference through chemical mapping and sequencing remains under question (3)(4)(5)(6)(7)(8). For example, models of the 9-kb HIV-1 RNA genome have been repeatedly revised with updates to the selective 2′-OH acylation by primer extension (SHAPE) protocol, data processing, and computational assumptions (2,(9)(10)(11), and the majority of this RNA's helices remain uncertain.…”

RNA structure through multidimensional chemical mapping

“…Chemical modification by DMS or other probes can be rapidly read out at every nucleotide of an RNA through primer extension reactions that terminate immediately 3′ to the modified bases, followed by electrophoresis or next-generation sequencing of the resulting cDNA products. The currently available set of chemical and enzymatic probes of RNA structure and methodological accelerations have been described in several recent reviews (Eddy, 2014;Kwok et al 2015;Weeks, 2010) and these methods continue to be advanced (see, e.g. Kielpinski & Vinther, 2014;Poulsen et al 2015;Spitale et al 2015). )…”

“…For example, reanalysis of a model based on selective 2´-OH acylation by primer extension (SHAPE) of the 9173-nucleotide HIV-1 RNA genome (Watts et al 2009) suggested that more than half of the presented helices were not well-determined (Kladwang et al 2011c), and subsequent work, including both experimental and computational improvements, have significantly revised these uncertain regions (Pollom et al 2013;Siegfried et al 2014;Sukosd et al 2015). The debate over whether these methods produce acceptable structure accuracies continues (Deigan et al 2009;Eddy, 2014;Kladwang et al 2011c;Leonard et al 2013;Rice et al 2014;Sukosd et al 2013;Tian et al 2014) and will not be reviewed in detail here. There is general agreement, however, on some points.…”

“…(Bothe et al 2011;Filbin & Kieft, 2009;Schneemann, 2006) Rouskin et al 2014;Spitale et al 2015)]. On the other hand, whether these conformational changes are functional or simply 'structural noise' is unknown for most regions, and the uncertainty is exacerbated by the difficulty of deconvolving the component structures from data that average over the entire ensemble of structures (Eddy, 2014;Washietl et al 2012). MCM measurements give rich data on RNA structure and, in favorable cases, allow deconvolution of ensembles of secondary and tertiary structures from experiments.…”

“…RNA's biological roles include catalysis of peptide bond formation and deciphering the genetic code in all living systems; elaborate alternative splicing of RNA messages in different tissues during metazoan development and evolution; and packaging, replication, and processing of pervasive parasitic elements, including viruses and retrotransposons [see (Gesteland et al 2006) and references therein]. Even as the RNAs involved in these processes have been under intense investigation, a vast number of additional RNA molecules are being discovered in genomic segments that do not code for proteins but appear to be transcribed and processed in a regulated manner (see Amaral et al 2008;Eddy, 2014;Qureshi & Mehler, 2012 and references therein). Understanding whether, when, and how these RNA molecules functionally impact complex organisms is a major current challenge in biology.…”