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

Ryan Damme, Kongpan Li, Minjie Zhang, Jianhui Bai, Wilson H. Lee, Joseph D. Yesselman, Zhipeng Lu () and Willem A. Velema ()
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Ryan Damme: University of Southern California
Kongpan Li: University of Southern California
Minjie Zhang: University of Southern California
Jianhui Bai: University of Southern California
Wilson H. Lee: University of Southern California
Joseph D. Yesselman: University of Nebraska-Lincoln
Zhipeng Lu: University of Southern California
Willem A. Velema: Radboud University Nijmegen

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-28602-3

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