The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest

Cell. 2019 Jan 10;176(1-2):391-403.e19. doi: 10.1016/j.cell.2018.11.004. Epub 2018 Dec 6.

Abstract

Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here, we introduce XRNAX, a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs) and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-related changes in the total proteome. In addition, we combine XRNAX with crosslinking immunoprecipitation sequencing (CLIP-seq) to validate the interaction of ncRNA with lamin B1 and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.

Keywords: RNA-binding domain; Trizol; UV-crosslinking; autophagy; mRNA; mass spectrometry; ncRNA; protein-RNA interactions; proteomics; ribophagy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Exosome Multienzyme Ribonuclease Complex / metabolism
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Immunoprecipitation / methods
  • Lamin Type B / metabolism
  • Protein Binding / genetics
  • Protein Binding / physiology
  • Protein Biosynthesis / genetics
  • Protein Biosynthesis / physiology
  • Protein Processing, Post-Translational
  • Proteins / isolation & purification
  • Proteins / metabolism
  • Proteome / metabolism
  • Proteomics / methods
  • RNA / genetics
  • RNA / isolation & purification*
  • RNA / metabolism
  • RNA, Messenger / metabolism
  • RNA, Untranslated / metabolism
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism
  • Transcriptome

Substances

  • EXOSC2 protein, human
  • Lamin Type B
  • Proteins
  • Proteome
  • RNA, Messenger
  • RNA, Untranslated
  • RNA-Binding Proteins
  • lamin B1
  • RNA
  • Exosome Multienzyme Ribonuclease Complex