The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.
The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.
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DOI：
10.1371/journal.pone.0088190
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Matzke M
中科院分区：
文献类型：
--
作者：
Sasaki T;Lorković ZJ;Liang SC;Matzke AJ;Matzke M
RDM 1（RNA-Directed DNA Methylation 1）是一种小分子植物特异性蛋白，是RNA指导的DNA甲基化（RdDM）所必需的。RDM 1与RNA聚合酶II（Pol II）、Argonaute 4（AGO 4）和从头DNA甲基转移酶结构域重排甲基转移酶2（DRM 2）相互作用，并与甲基化单链DNA结合。作为迄今为止鉴定的唯一与DRM 2直接相互作用的蛋白质，RDM 1通过将从头DNA甲基转移酶活性与AGO 4连接而在RdDM机制中起关键作用，AGO 4结合短干扰RNA（siRNA），其推测与在靶位点合成的Pol II或Pol V支架转录物碱基配对。RDM 1还与染色质重塑剂DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1（DRD 1）和染色体结构维持单铰链蛋白DEFECTIVE IN MERISTEM SILENCING 3（DMS 3）一起作用，形成DDR复合物，促进Pol V支架转录物的合成。目前尚不清楚快速反应机制1在复员方案综合体中以及作为连接快速反应机制2和AGO 4的一个因素发挥作用的方式。RDM 1不含已知的蛋白质结构域，但先前的结构分析表明，不同的区域分别产生疏水口袋和促进同源二聚体形成。我们已经测试了在预测的口袋和二聚化区域中改变的RDM 1的几种突变形式，其能够补充RdDM和转录基因沉默中的缺陷，支持Pol V转录物的合成，形成同源二聚体，并与DMS 3相互作用。我们的研究结果表明，形成同源二聚体的能力是必不可少的RDM 1在RdDM通路中充分发挥作用，可能是特别重要的从头甲基化步骤。
RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step.
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