Riboproteomics: A versatile approach for the identification of host protein interaction network in plant pathogenic noncoding RNAs

PLoS One. 2017 Oct 26;12(10):e0186703. doi: 10.1371/journal.pone.0186703. eCollection 2017.

Abstract

Pathogenic or non-pathogenic small (17 to 30 nt) and long (>200 nt) non-coding RNAs (ncRNAs) have been implicated in the regulation of gene expression at transcriptional, post-transcriptional and epigenetic level by interacting with host proteins. However, lack of suitable experimental system precludes the identification and evaluation of the functional significance of host proteins interacting with ncRNAs. In this study, we present a first report on the application of riboproteomics to identify host proteins interacting with small, highly pathogenic, noncoding satellite RNA (sat-RNA) associated with Cucumber mosaic virus, the helper virus (HV). RNA affinity beads containing sat-RNA transcripts of (+) or (-)-sense covalently coupled to cyanogen bromide activated sepharose beads were incubated with total protein extracts from either healthy or HV-infected Nicotiana benthamiana leaves. RNA-protein complexes bound to the beads were eluted and subjected to MudPIT analysis. Bioinformatics programs PANTHER classification and WoLF-PSORT were used to further classify the identified host proteins in each case based on their functionality and subcellular distribution. Finally, we observed that the host protein network interacting with plus and minus-strand transcripts of sat-RNA, in the presence or absence of HV is distinct, and the global interactome of host proteins interacting with satRNA in either of the orientations is very different.

MeSH terms

  • Cucumovirus / genetics
  • Cucumovirus / metabolism*
  • Helper Viruses / genetics
  • Helper Viruses / metabolism*
  • Nicotiana / genetics
  • Nicotiana / metabolism*
  • Nicotiana / virology
  • Plant Proteins / classification
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Proteomics / methods
  • RNA, Untranslated / classification
  • RNA, Untranslated / genetics
  • RNA, Untranslated / metabolism*
  • RNA, Viral / classification
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*

Substances

  • Plant Proteins
  • RNA, Untranslated
  • RNA, Viral

Grants and funding

This study was partly funded by a grant from University of California Agricultural Experimental Station Mission Funding Program (19900). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.