dsRNA with 5' overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

EMBO J. 2009 Mar 4;28(5):545-55. doi: 10.1038/emboj.2009.2. Epub 2009 Jan 22.

Abstract

In plants, SGS3 and RNA-dependent RNA polymerase 6 (RDR6) are required to convert single- to double-stranded RNA (dsRNA) in the innate RNAi-based antiviral response and to produce both exogenous and endogenous short-interfering RNAs. Although a role for RDR6-catalysed RNA-dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA-binding protein with unexpected substrate selectivity favouring 5'-overhang-containing dsRNA. The conserved XS and coiled-coil domains are responsible for RNA-binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi-based host immune response, is a dsRNA-binding protein with similar specificity to SGS3. In competition-binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5' overhang is required for subsequent steps in RNA-mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.

Publication types

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

MeSH terms

  • 5' Flanking Region
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Begomovirus / metabolism
  • Binding, Competitive
  • Electrophoretic Mobility Shift Assay
  • Lycopersicon esculentum / metabolism
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Point Mutation
  • Protein Binding
  • Protein Multimerization
  • RNA Interference / physiology*
  • RNA Replicase / genetics
  • RNA Replicase / metabolism*
  • RNA, Double-Stranded / metabolism*
  • RNA-Binding Proteins / metabolism
  • Substrate Specificity
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Arabidopsis Proteins
  • Plant Proteins
  • RNA, Double-Stranded
  • RNA-Binding Proteins
  • SGS3 protein, Arabidopsis
  • Viral Proteins
  • RDR6 protein, Arabidopsis
  • RNA Replicase