RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure

Abstract

SUMMARY RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here we develop PARIS, a method based on reversible psoralen-crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher order architectures, and RNA:RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures, and discovered conserved long-range and alternative structures. XIST, a lncRNA essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome.

A mutually exclusive stem–loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila

“…Using reversible AMT cross-linking, a recent study identified many previously unknown RNA-RNA contacts in human and mouse cells (Lu et al 2016), mostly in the form of duplexes. Interestingly, this study also uncovered several putative alternating structures, similar to what we observed in our study of roX RNAs.…”

Genome-Wide Analysis of RNA Secondary Structure

“…Likewise, a reagent that could distinguish between protections arising from protein binding and those arising from base pairing would yield a quantum advance by providing comprehensive data on base pairing along transcripts and across transcriptomes. Some progress has been made very recently in this direction (3,70,96). Such probes would help determine the extent to which the above-mentioned discrepancies regarding in vivo versus in vitro protections that have arisen in structure-probing studies are the result of protein binding in vivo or of RNA refolding in nonbiological test-tube conditions.…”

RNA structure inference through chemical mapping after accidental or intentional mutations

“…Even for small RNA domains, SHAPE and dimethyl sulfate (DMS) (methylation of N1 and N3 atoms at A and C) have produced misleading secondary structures for ribosomal domains and blind modeling challenges that have been falsified through crystallography or mutagenesis (3,7,12,13). In alternative approaches based on photoactivated cross-linkers, many helix detections appear to be false positives, based on ribosome data in vitro and in vivo (14,15).…”

“…Even for small RNA domains, SHAPE and dimethyl sulfate (DMS) (methylation of N1 and N3 atoms at A and C) have produced misleading secondary structures for ribosomal domains and blind modeling challenges that have been falsified through crystallography or mutagenesis (3,7,12,13). In alternative approaches based on photoactivated cross-linkers, many helix detections appear to be false positives, based on ribosome data in vitro and in vivo (14,15).The confidence and structural accuracy of chemical-mapping methods can be improved by applying perturbations to the RNA sequence before chemical modification. In the mutate-and-map strategy, mapping not just the target RNA sequence but also a comprehensive library of point mutants reveals which nucleotides respond to perturbations at every other nucleotide, enabling direct inference of pairs of residues that interact to form structure (16,17).…”