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Published January 18, 2024 | Version v1
Journal article Open

KARR-seq reveals cellular higher-order RNA structures and RNA–RNA interactions

Creators

  • 1. University of Chicago
  • 2. Agency for Science, Technology and Research
  • 3. The Ohio State University
  • 4. University of Massachusetts, Amherst

Description

RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA–RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA–RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA–RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.

Data availability

Public sequencing data used in this study were acquired from the Gene Expression Omnibus (GEO) with accession numbers as follows: GSE127188 (RIC-seq, HeLa); GSE74353 (PARIS, HEK293T); GSE138058 (G3BP1-APEX-seq); and GSE121952 (ribosome profiling). Cryo-EM structures were acquired from the Protein Data Bank (accession codes: 7QXB for TERC, 6QX9 for U3 and 4V6X for 18S and 28S). RBP eCLIP BAM files were accessed from https://www.encodeproject.org/. Raw and analyzed data for all sequencing experiments have been deposited at the GEO (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE166155.

The source code of KARR-seq is freely available at https://github.com/ouyang-lab/KARR-seq.

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KARR-seq-reveal-cellular-higher-order-RNA-structures-and-RNA–RNA-interactions.pdf

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Additional details

Identifiers

DOI
10.1038/s41587-023-02109-8
Other
oai:uchicago.tind.io:10623

Funding

National Institutes of Health
R01HG012780
National Institutes of Health
RM1HG008935
National Institutes of Health
R35GM124998

UChicago Information

Division(s)
Physical Sciences Division
Department(s)
Biochemistry and Molecular Biology, Chemistry
Center(s) or Institute(s)
Institute for Biophysical Dynamics