Identification of proteins and miRNAs that specifically bind an mRNA in vivo

Nat Commun. 2019 Sep 16;10(1):4205. doi: 10.1038/s41467-019-12050-7.

Abstract

Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / genetics
  • Caenorhabditis elegans / metabolism*
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Protein Binding
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Caenorhabditis elegans Proteins
  • DAZ-1 protein, C elegans
  • GLD-1 protein, C elegans
  • LIN-41 protein, C elegans
  • MIRN84 microRNA, C elegans
  • MicroRNAs
  • RNA, Messenger
  • RNA-Binding Proteins
  • Transcription Factors