In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
Kyungmin Baeg,
Yukihide Tomari,
Hiro-oki Iwakawa
Affiliations
Kyungmin Baeg
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, JapanDepartment of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Yukihide Tomari
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, JapanDepartment of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Hiro-oki Iwakawa
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, JapanDepartment of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.
