title: Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus.
abstract: Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III-like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2'-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg(2+) was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn(2+). Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.
authors.affiliation: AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, Neustadt a.d.W., Germany.|Department of Agricultural Sciences, PO Box 27, 00014 University of Helsinki, Helsinki, Finland.
authors.forename: Isabel
authors.initials: I
authors.lastname: Weinheimer
authors.affiliation: AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, Neustadt a.d.W., Germany.
authors.forename: Kajohn
authors.initials: K
authors.lastname: Boonrod
authors.affiliation: AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, Neustadt a.d.W., Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Moser
authors.affiliation: Centre for Organismal Studies (COS) Heidelberg, Im Neuenheimer Feld 230, Heidelberg, Germany.|AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, Neustadt a.d.W., Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wassenegger
authors.affiliation: AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, Neustadt a.d.W., Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Krczal
authors.affiliation: Institute of Biotechnology, PO Box 65, 00014 University of Helsinki, Helsinki, Finland.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Butcher
authors.affiliation: Department of Agricultural Sciences, PO Box 27, 00014 University of Helsinki, Helsinki, Finland.
authors.forename: Jari P T
authors.initials: JPT
authors.lastname: Valkonen
mesh_terms: D002413:Cations, Divalent / Q000378:metabolism; D029383:Crinivirus / Q000201:enzymology*; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D027723:Ipomoea batatas / Q000821:virology; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology; D011485:Protein Binding; D055503:Protein Multimerization; D012330:RNA, Double-Stranded / Q000378:metabolism*; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D012330:RNA, Double-Stranded; D014764:Viral Proteins; D037521:Virulence Factors; D008345:Manganese; D043244:Ribonuclease III; D008274:Magnesium