Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells

Abstract

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2′-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

Classification and clustering of RNA crosslink-ligation data reveal complex structures and homodimers

“…To further validate the U2 alternative conformations, we used an orthogonal crosslinking method, Selective 2′-Hydroxyl Acylation Reversible Crosslinking (SHARC) ( Van Damme et al 2022 ). SHARC reagents crosslink RNA nucleotides in spatial proximity (not base-pairing) at the 2′-OH positions, and the crosslinking is reversible by mild alkaline hydrolysis.…”

Chemical RNA Crosslinking: Mechanisms, Computational Analysis and Biological Applications

“…16,17 More recent bifunctional acylators target the 2'-OH position to establish crosslinking. [39][40][41] The unusual nucleophilicity of the 2'-OH position has been attributed to oxyanion formation at this position due to inductive effects provided by neighboring 3' and 4' oxygens and the nucleobase nitrogen. 42,43 Pyrimidine bases can undergo [2+2] photocycloadditions using their C5-C6 double bond (blue, Fig.…”

“…In 2022, Lu, Velema and colleagues explored the possibility to exploit bifunctional acylators for RNA structure determination. 40 This method, named Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), used simple dicarboxylic acids that could be activated with CDI in a single step to crosslink RNA. In particular, DPI (Fig.…”

“…106 Proteinase treatment after crosslinking can exclude the protein-mediated proximities. 40,57 Careful comparison of data from different types of crosslinkers will provide deeper insights into the organization of the RNP complexes. Sequencing reads from crosslink experiments can be aligned to the reference using various types of mappers, such as STAR and Bowtie.…”

“…This approach was first proposed in 2016 and has been systematically benchmarked, optimized and widely applied to various experimental methods. 33,40,60,97,108 The clustering of multiple different DGs provides direct evidence for the existence of alternative or dynamic conformations. Such conformations are easily detected for psoralen crosslinked RNA fragments, given the mutually exclusive base pairing (except the triplexes and G-quadruplexes where each stretch of nucleotides can participate in multiple consecutive contacts).…”