RNA指导的DNA甲基化中Pol V最大亚基CTD的功能剖析
Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation.
作者信息
Wendte Jered M, Haag Jeremy R, Singh Jasleen, McKinlay Anastasia, Pontes Olga M, Pikaard Craig S
机构信息
Department of Biology, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA.
Department of Biology, Indiana University, 915 East Third Street, Bloomington, IN 47405, USA; Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63130, USA.
出版信息
Cell Rep. 2017 Jun 27;19(13):2796-2808. doi: 10.1016/j.celrep.2017.05.091.
Plant multisubunit RNA polymerase V (Pol V) transcription recruits Argonaute-small interfering RNA (siRNA) complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive C-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro yet essential in vivo. One CTD subdomain (DeCL) is required for Pol V function at virtually all loci. Other CTD subdomains have locus-specific effects. In a yeast two-hybrid screen, the 3'→ 5' exoribonuclease RRP6L1 was identified as an interactor with the DeCL and glutamine-serine (QS)-rich subdomains located downstream of an Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3' ends of Pol V transcripts sliced by Argonaute 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.
植物多亚基RNA聚合酶V（Pol V）转录招募AGO（Argonaute）-小干扰RNA（siRNA）复合体，这些复合体决定了用于基因沉默的RNA指导的DNA甲基化（RdDM）位点。Pol V的最大亚基NRPE1由Pol II的最大亚基进化而来，但具有独特的C端结构域（CTD）。我们发现，Pol V的CTD在体外对催化活性可有可无，但在体内却是必需的。几乎所有位点的Pol V功能都需要一个CTD亚结构域（DeCL）。其他CTD亚结构域具有位点特异性效应。在酵母双杂交筛选中，3'→5'外切核糖核酸酶RRP6L1被鉴定为与位于AGO结合亚结构域下游的DeCL和富含谷氨酰胺-丝氨酸（QS）的亚结构域相互作用的蛋白。实验证据表明，RRP6L1修剪由AGO4切割的Pol V转录本的3'末端，这提示了一种模型：CTD能够使AGO4和RRP6L1的RNA加工活动在空间和时间上协调。
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