Curaba Julien, Chen Xuemei
Department of Botany and Plant Sciences, University of California, Riverside, California 92521.
Department of Botany and Plant Sciences, University of California, Riverside, California 92521.
J Biol Chem. 2008 Feb 8;283(6):3059-3066. doi: 10.1074/jbc.M708983200. Epub 2007 Dec 6.
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.
在拟南芥中，遗传学证据表明，RNA依赖性RNA聚合酶6（RDR6）在至少四种RNA沉默途径中发挥着重要作用，这些途径的功能范围从抵御转基因或病毒到发育和应激反应中的内源基因调控。尽管RDR6在RNA沉默中起关键作用，但其生化活性尚未得到表征。在本研究中，我们在本氏烟草中瞬时表达了拟南芥RDR6，并在体外研究了免疫纯化的RDR6的生化活性。我们发现RDR6具有末端核苷酸转移酶活性以及对单链RNA的不依赖引物的RNA聚合酶活性。我们发现RDR6无法区分有无帽或聚（A）尾的RNA。我们还证明RDR6对单链DNA具有很强的聚合酶活性。所有这些活性都需要保守的催化Asp（867）残基。我们的发现对体内涉及RDR6的过程具有重要意义，并为拟南芥中RNA沉默的机制提供了新的生化见解。
