Studying RNA-Protein Interaction Using Riboproteomics

Methods Mol Biol. 2021;2170:213-218. doi: 10.1007/978-1-0716-0743-5_15.

Abstract

Protein-protein interactions (PPI) are vital in regulating the biological and physiological functions in a given cell or organism. Proteomics, in conjunction with bioinformatic tools, represents the study involving the characterization of the protein content of the genome of a given biological system. Like PPI, an interaction between either coding or noncoding RNA and a complex set of host proteins protein plays an essential role in gene expression at translational, posttranscriptional, and epigenetic level. Although a wide range of techniques such as shotgun proteomics, MuDPIT, etc. are available for characterizing PII, those for characterizing RNA-protein interactions are infancy. Given the significance of the long noncoding RNAs (lnc-RNA) in plant biology, it is imperative to isolate and characterize the functionality of the host proteome interacting with RNA. In this context, riboproteomics approach becomes a valuable tool to study these interactions. Here, using a noncoding plant pathogenic satellite-RNA (Sat-RNA) of Cucumber mosaic virus (CMV) as an RNA source, we describe a stepwise protocol for identifying the host proteome interacting specifically with the Sat-RNA. This protocol streamlines steps starting from in vitro transcription of RNA, preparation of RNA affinity column, preparation of cell lysate from Nicotiana benthamiana leaves infected with the Sat-RNA followed by the Co-IP and preparation of samples for LC-MS/MS. We believe this approach is applicable to a wide range of RNAs of any nature associated with eukaryotic and prokaryotic organisms.

Keywords: Cucumber mosaic virus; LC-MS/MS; Long noncoding RNA; Proteomics; RNA viruses; Riboproteomics; Satellite-RNA.

MeSH terms

  • Chromatography, Liquid
  • Cucumovirus
  • Plant Diseases / virology
  • Plant Leaves / virology
  • Protein Binding
  • Proteomics
  • RNA, Untranslated / genetics
  • RNA, Untranslated / metabolism*
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • Tandem Mass Spectrometry / methods*
  • Tobacco / virology

Substances

  • RNA, Untranslated
  • RNA, Viral