title: dsRNA with 5' overhangs contributes to endogenous and antiviral RNA silencing pathways in plants.
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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Ryuya
authors.initials: R
authors.lastname: Fukunaga
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D024506:5' Flanking Region; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D053839:Begomovirus / Q000378:metabolism; D001667:Binding, Competitive; D024202:Electrophoretic Mobility Shift Assay; D018551:Solanum lycopersicum / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C410507:SGS3 protein, Arabidopsis; D014764:Viral Proteins; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase