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

Minjie Zhang,Jian-Fu Chen,Willem A Velema,Kongpan Li,Chengqing Yu,Wilson H Lee,Maia L Corpuz,Zhipeng Lu,Ryan Van Damme,Jianhui Bai

doi:10.1038/s41467-021-22552-y

Minjie Zhang, Jian-Fu Chen + Show 8 more

Open Access

https://doi.org/10.1038/s41467-021-22552-y

Copy DOI

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.

Highlights

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states
Base pair stacking is the dominant force in RNA structures and RNA–RNA interactions; direct determination of base pairs is a critical step toward decoding the structural basis of RNA-mediated regulation in cells
We discover a network of dynamic RNA structures and interactions in the U8 snoRNA involved in ribosomal RNA processing

Summary

Introduction

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. We and others developed approaches to determine RNA base pairs, based on the principle of crosslinking, proximity ligation, and high-throughput sequencing[4,5,6,7,8,9] These methods, including PARIS (psoralen analysis of RNA interactions and structures), SPLASH, LIGR-seq, and COMRADES, allowed direct analysis of RNA duplexes at the transcriptome level, achieving single-molecule accuracy and near base pair resolution. We introduce chemical and enzymatic approaches to prevent and bypass photochemical damages to RNA, a fundamental problem in RNA research Together, these optimizations in PARIS2 resulted in >4000-fold increased efficiency, and importantly, the deep mechanistic insights into photochemistry, RNA chemistry, and enzymology for individual improvements are broadly applicable in RNA studies. The PARIS2 method will enable more rapid and facile analysis of structural basis of RNA functions in various biological systems and human diseases

Methods

Results

Conclusion