Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

PLoS One. 2015 Mar 20;10(3):e0120100. doi: 10.1371/journal.pone.0120100. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / enzymology*
  • Arabidopsis / genetics*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Gene Expression Regulation, Plant
  • RNA, Double-Stranded / biosynthesis*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism*

Substances

  • Arabidopsis Proteins
  • RNA, Double-Stranded
  • RDR2 protein, Arabidopsis
  • RDR6 protein, Arabidopsis
  • RNA-Dependent RNA Polymerase

Grants and funding

This research was supported by a grant from Agence Nationale de la Recherche, ANR-06-BLAN-0256 (to N.F., B.C. and P.C). A.D and M.F were supported by a PhD fellowship from the Ecole Doctorale des Sciences de la Vie et de la Santé, Aix-Marseille Université, Ministère de l’enseignement supérieur et de la recherche. Funding for open access charge: Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Aix-Marseille Université. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.