A novel requirement for Drosha in maintenance of mammalian CG methylation

Using a protein interaction screen, we identify the Microprocessor component Drosha as a novel Dnmt1-interactor. Drosha-deficient ES cells display genomic hypomethylation which is not accounted for by changes in the levels of Dnmt proteins. Both genetic and transfection studies show that Drosha stimulates Dnmt1 methyltransferase activity. We identify two transcripts that are specifically upregulated in Drosha but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight Drosha as a novel regulator of mammalian DNA methylation and we propose that Drosha-mediated processing of RNA is necessary to ensure full Dnmt1 activity. This adds to the Drosha repertoire of non-miRNA dependent functions as well as implicating RNA in regulating Dnmt1 activity and correct levels of genomic methylation. Mapping of 5-methylcytosine in wild type and Drosha-deficient ES cells by MethylC-Seq and mapping of RNA in wild type and Drosha-deficient ES cells by RNA-Seq

本研究通过蛋白质相互作用筛选，将微处理器复合体（Microprocessor）的组分Drosha鉴定为一种新型DNA甲基转移酶1（Dnmt1）互作蛋白。Drosha缺陷型胚胎干细胞（ES cells）表现出基因组低甲基化表型，且该表型无法由Dnmt家族蛋白的水平变化所解释。遗传学实验与转染实验均证实，Drosha可促进Dnmt1的甲基转移酶活性。本研究鉴定出两类转录本，其表达仅在Drosha缺陷型胚胎干细胞中特异性上调，而在Dicer缺陷型胚胎干细胞中无此变化。这些转录本中预测可形成茎环结构的区域可被微处理器复合体加工，且在体外能够抑制DNMT1介导的甲基化反应。本研究结果将Drosha确立为哺乳动物DNA甲基化的新型调控因子，并提出Drosha介导的RNA加工过程是维持Dnmt1完整活性所必需的。这一发现拓展了Drosha不依赖miRNA的功能谱系，同时也揭示了RNA在调控Dnmt1活性及维持基因组甲基化水平稳态中的关键作用。本研究分别通过MethylC测序（MethylC-Seq）与RNA测序（RNA-Seq），绘制了野生型与Drosha缺陷型胚胎干细胞的5-甲基胞嘧啶图谱与RNA表达图谱。