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

Ryan Van Damme,Jianhui Bai,Willem A Velema,Joseph D Yesselman,Zhipeng Lu,Minjie Zhang,Kongpan Li,Wilson H Lee

doi:10.1038/s41467-022-28602-3

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.

Highlights

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes
Together with the alternative conformations in 7SL (Supplementary Fig. 8), these results suggest that Spatial 2′-Hydroxyl Acylation Reversible Crosslinking (SHARC)-exo captures static and dynamic structures in cells
This study reports a series of reversible crosslinkers, SHARC, that can capture spatial proximity in RNA with high efficiency

Summary

Introduction

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. 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. Correlated chemical probing methods such as multiplexed OH cleavage analysis (MOHCA), mutate-and-map (M2), and RNA interacting group mutational profiling (RING-MaP) infer spatial proximity of nucleotides but provides fuzzy distances to constrain 3D modeling[14,15,16,17,18]. The integration of SHARC crosslinking, exo trimming, proximity ligation, and high throughput sequencing (SHARC-exo) enables transcriptomewide analysis of spatial distances between nucleotides at nanometer resolution in cells, without sequence length limitations. We rigorously benchmarked the distance measurement and structure capture using complex, yet well-studied models in cells, such as the ribosome, spliceosome, 7SL, and RNase P, revealing both static structures, interactions, and alternative conformations

Methods

Results

Conclusion