Pol IV 大亚基 CTD 定量影响指导 RNA 导向 DNA 甲基化的 siRNA 水平。

The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation.

机构信息

Department of Biology, Indiana University, 915 E. Third Street, Bloomington, IN 47405, USA.

Division of Biology and Biomedical Sciences, Washington University, St Louis, MO 63130, USA.

出版信息

Nucleic Acids Res. 2019 Sep 26;47(17):9024-9036. doi: 10.1093/nar/gkz615.

DOI:10.1093/nar/gkz615

PMID:31329950

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6753486/

Abstract

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV's largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

摘要

在植物中，核多亚基 RNA 聚合酶 IV 和 V 是与 RNA 聚合酶 II 相关的酶，它们合成非编码 RNA，用于 RNA 指导的 DNA 甲基化 (RdDM) 和转录基因沉默。在这里，我们测试了 Pol IV 最大亚基的 C 末端结构域 (CTD) 的重要性，因为 Pol II CTD 介导了 Pol II 转录的多个方面。我们表明，CTD 对于 Pol IV 的催化活性和 Pol IV 终止依赖性激活 RNA 依赖性 RNA 聚合酶 2 是可有可无的，Pol IV 与 Pol II 合作生成 24nt siRNA 的 dsRNA 前体，该 siRNA 指导 RdDM。然而，当 CTD 缺失时，24nt siRNA 水平下降约 80%。依赖 RNA 的胞嘧啶甲基化也减少，但仅减少约 20%，表明 siRNA 水平通常超过大多数靶位点甲基化所需的水平。失去 CTD 后受 Pol IV 影响的基因座主要位于染色体臂上，类似于依赖 CLSY1/2 或 SHH1 的基因座，CLSY1/2 或 SHH1 是涉及 Pol IV 募集的蛋白质。然而，CTD 的缺失并没有表现出与 clsy 或 shh1 突变体相同的表型，这与 CTD 影响靶基因座 Pol IV 活性的招募后方面一致。