High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

Nat Commun. 2018 Oct 4;9(1):4084. doi: 10.1038/s41467-018-06207-z.

Abstract

Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Escherichia coli
  • High-Throughput Screening Assays / methods*
  • RNA, Bacterial / metabolism*
  • Regulatory Sequences, Ribonucleic Acid*

Substances

  • RNA, Bacterial
  • Regulatory Sequences, Ribonucleic Acid