Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, F-67084 Strasbourg, France.
Howard Hughes Medical Institute, Indiana University, Bloomington, IN 47405, USA.
Nucleic Acids Res. 2019 Sep 26;47(17):9037-9052. doi: 10.1093/nar/gkz618.
RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.
RNA 引导的监测系统限制了转座元件 (TEs) 在宿主基因组中的活性。在植物中，RNA 聚合酶 IV (Pol IV) 将 TEs 转录成初级转录本，RDR2 从这些转录本中合成双链 RNA 前体，用于引导 TE 甲基化和沉默的小干扰 RNA (siRNAs)。Pol IV 的核心亚基，即 RNA 聚合酶 II 亚基的同源物，是如何分化以支持富含 TE、抑制性染色质环境中的 siRNA 生物发生的，目前还不太清楚。在这里，我们研究了 Pol IV 最大亚基 NRPD1 的 N 端。该 N 端带有错义突变的拟南芥系产生野生型 (WT) 水平的 NRPD1，其与其他 Pol IV 亚基和 RDR2 共纯化。我们的体外转录和基因组分析表明，NRPD1 N 端对于强大的 Pol IV 依赖性转录、siRNA 产生和 DNA 甲基化至关重要。然而，在一个突变基因型中观察到的残留 RNA 指导的 DNA 甲基化表明，Pol IV 可以在通常在 WT 植物中观察到的高水平 siRNA 下不依赖地发挥作用。这种突变破坏了 Pol IV 中独特保守的一个基序，削弱了该酶抑制反转录转座子动员的能力。我们提出，NRPD1 N 端基序的进化是为了调节 Pol IV 在基因组监测中的功能。
