title: Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.
abstract: Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants.
authors: Aix-Marseille Université, Laboratoire de Génétique et Biophysique des Plantes, Marseille, France; Centre National de la Recherche Scientifique, UMR 7265, Biologie Végétale et Microbiologie Environnementale, Marseille, France; Commissariat à l'Énergie Atomique, Département des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologies, Marseille, France.
authors: Anthony
authors: A
authors: Devert
authors: Aix-Marseille Université, Laboratoire de Génétique et Biophysique des Plantes, Marseille, France; Centre National de la Recherche Scientifique, UMR 7265, Biologie Végétale et Microbiologie Environnementale, Marseille, France; Commissariat à l'Énergie Atomique, Département des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologies, Marseille, France.
authors: Nicolas
authors: N
authors: Fabre
authors: Aix-Marseille Université, Laboratoire de Génétique et Biophysique des Plantes, Marseille, France; Centre National de la Recherche Scientifique, UMR 7265, Biologie Végétale et Microbiologie Environnementale, Marseille, France; Commissariat à l'Énergie Atomique, Département des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologies, Marseille, France.
authors: Maïna
authors: M
authors: Floris
authors: Aix-Marseille Université, AFMB UMR 7257, Marseille, France; CNRS, AFMB UMR 7257, Marseille, France.
authors: Bruno
authors: B
authors: Canard
authors: Aix-Marseille Université, Laboratoire de Génétique et Biophysique des Plantes, Marseille, France; Centre National de la Recherche Scientifique, UMR 7265, Biologie Végétale et Microbiologie Environnementale, Marseille, France; Commissariat à l'Énergie Atomique, Département des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologies, Marseille, France.
authors: Christophe
authors: C
authors: Robaglia
authors: Aix-Marseille Université, Laboratoire de Génétique et Biophysique des Plantes, Marseille, France; Centre National de la Recherche Scientifique, UMR 7265, Biologie Végétale et Microbiologie Environnementale, Marseille, France; Commissariat à l'Énergie Atomique, Département des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologies, Marseille, France.
authors: Patrice
authors: P
authors: Crété
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D012330:RNA, Double-Stranded / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D012330:RNA, Double-Stranded; C519655:RDR2 protein, Arabidopsis; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase