Biochemical Activities of Arabidopsis RNA-dependent RNA Polymerase 6

Julien Curaba,Xuemei Chen

doi:10.1074/jbc.m708983200

Abstract

In Arabidopsis, genetic evidence demonstrates that RNA-dependent RNA polymerase 6 (RDR6) plays a fundamental role in at least four RNA silencing pathways whose functions range from defense against transgenes or viruses to endogene regulation in development and in stress responses. Despite its critical role in RNA silencing, the biochemical activities of RDR6 have yet to be characterized. In this study, we transiently expressed Arabidopsis RDR6 in Nicotiana benthamiana and investigated the biochemical activities of immunopurified RDR6 in vitro. We showed that RDR6 possesses terminal nucleotidyltransferase activity as well as primer-independent RNA polymerase activity on single-stranded RNAs. We found that RDR6 cannot distinguish RNAs with or without a cap or poly(A) tail. We also demonstrated that RDR6 has strong polymerase activity on single-stranded DNA. All these activities require the conserved catalytic Asp(867) residue. Our findings have important implications on the processes involving RDR6 in vivo and provide new biochemical insights into the mechanisms of RNA silencing in Arabidopsis.

Highlights

In the model plant Arabidopsis, genetic evidence demonstrates the requirement for RNA-dependent RNA polymerase 6 (RDR6) in a number of silencing pathways [3, 4, 19, 21, 23, 25], but the RNA-dependent RNA polymerase activity of RDR6 has been inferred based on sequence homology with the tomato enzyme and has yet to be confirmed and characterized biochemically
Full-length RDR6 cDNA was cloned behind the cauliflower mosaic virus (CaMV) 35S promoter and in-frame with an N-terminal HA tag
Biochemical Properties of RDR6—Our studies demonstrate that RDR6 possesses RNA-dependent RNA polymerase activity on ssRNAs

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The full-length cDNA of RDR6 (3.6 kb) was amplified from the ATG to the stop codon by PCR using the primers R6-1 (5Ј-ccggaattcgagaaatggggtcagagggaaata-3Ј) and R6-2 (5Ј-ccgcggccgcaacataaccttttagagacgtgagc3Ј). Reactions were stopped by incubation at 65 °C for 10 min and were directly loaded on a 6% polyacrylamide, 7 M urea gel and the polyadenylated RNAs were excised from the gel and isolated. Reactions were stopped by adding 50 ␮l of buffer (20 mM Tris-HCl, pH 7.6, 20 mM MgCl2, 2% SDS), 90 ␮g of proteinase K (Fermentas) in a final volume of 100 ␮l, followed by incubation for 20 min at 65 °C. Assays for Priming Activity with an RNA Oligonucleotide—30 pmol of miR173* (5Ј-rGrArUrUrCrUrCrUrGrUrGrUrArArGrCrGrArArArG-3Ј) was mixed with 15 pmol of the 246R template in a 10-␮l solution containing 50 mM HEPES-KOH, pH 7.4, and 100 mM KCl. The mixture was incubated for 3 min at 95 °C and for 10 min at 42 °C, and used for RDR assay as previously described

RESULTS

When the reaction was performed in the presence of both cold

DISCUSSION