Biochemical activities of Arabidopsis RNA-dependent RNA polymerase 6
Biochemical activities of Arabidopsis RNA-dependent RNA polymerase 6
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DOI：
10.1074/jbc.m708983200
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发表时间：
2008-02-08
影响因子：
4.8
通讯作者：
Chen, Xuemei
中科院分区：
文献类型：
--
作者：
Curaba, Julien;Chen, Xuemei
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沉默途径中起着重要作用，其功能范围从防御转基因或病毒到发育和胁迫反应中的内源基因调控。尽管RDR6在RNA沉默中起着关键作用，但其生物化学活性尚未得到表征。在这项研究中，我们瞬时表达拟南芥RDR6在烟草本塞姆亚纳和研究免疫纯化的RDR6在体外的生化活性。我们发现，RDR6具有末端核苷酸转移酶活性以及引物非依赖性RNA聚合酶活性的单链RNA。我们发现RDR6不能区分有或没有帽或poly（A）尾的RNA。我们还证明了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性Asp（867）残基。我们的研究结果对RDR6在体内的表达过程具有重要意义，并为拟南芥RNA沉默机制提供了新的生化见解。
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.