Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases

Abstract

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (−) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.

Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells

“…We recovered 1.01%, 1.31% and 1.89% RNA fragments as crosslinked using 5, 12.5, or 25 mM DPI, respectively (Supplementary Fig. 3f-h, comparable to psoralens used in the PARIS method) 24 . PK digests proteins to <6 amino acids long 35 , and efficiently removed the vast majority of proteins (Supplementary Fig.…”

“…Crosslinked RNA samples are first digested with RNase III, which fragments both single and double-stranded RNA into short pieces 21 . RNA fragments are fractionated on a denatured-denatured 2-dimension (DD2D) gel 24 , where the second dimension is denser than the first (e.g., 16% vs. 8%). The differential gel densities enable the separation of crosslinked from non-crosslinked fragments.…”

“…Crosslinking and proximity ligation represents an alternative strategy to capture spatial distances among nucleotides, overcoming the limitations of correlated chemical probing 19 . Recently developed psoralen-crosslinking-based methods, such as PARIS, LIGR-seq, SPLASH, and COMRADES directly capture base pairs either within or between different RNA molecules in high throughput [20][21][22][23][24] . Psoralen crosslinks staggering pyrimidines in opposite strands through [2+2] photocycloadditions.…”

Rhinovirus Inhibitors: Including a New Target, the Viral RNA

“…RNA-binding compounds have been present in the virologists’ toolbox for a long time: (i) acridine orange and neutral red are well known from early studies on the uncoating of several Enteroviruses [ 186 , 187 ], (ii) SYTO 82 and RiboGreen were mainly used to follow RNA release from virions in live-cell microscopy and capillary electrophoresis [ 188 , 189 ], and (iii) psoralen analogues were employed in RNA interactome mapping in the Zika virus, SARS-CoV-2, enterovirus D68 [ 190 , 191 , 192 ], and in RV-A2 for covalently cross-linking double-stranded regions in the RNA [ 176 ]. RNA was also labelled inside RV-B14 with N-acetyl-aziridine [ 193 ].…”

“…Recently, short- and long-range interactions were demonstrated for the genomic RNA of enterovirus D68 [ 192 ] and other RNA viruses [ 191 , 217 ]. Such long-range interactions are probably involved in maintaining the spherical outline of the deproteinated viral RNA.…”