The role of the Xist 5’ m6A region and RBM15 in X chromosome inactivation

X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5’ end of the transcript, and also further 3’, in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist–mediated gene silencing. We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5’ m6A region in interspecific XX mouse embryonic stem cells (mESC). Following induction of Xist RNA expression we assay chromosome silencing using allelic RNA-seq and Xist m6A patterns using m6A-seq. Additionally we use Xist RNA FISH to analyse the effect of deleting the 5’ m6A region on the function of the endogenous Xist promoter. Purifying epitope tagged RBM15, we employ MS/MS analysis to define the RBM15 interactome in mESCs. We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing, and that the 5’ m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5’ m6A region without affecting the modification in exon VII. We show that in mESCs RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism哺乳动物X染色体失活的调控依赖于非编码（non-coding, nc）RNA Xist，该RNA可抑制其自身转录所在染色体的基因活性。Xist外显子I中靠近转录本5'端的区域，以及Xist外显子VII中更远的3'端区域，均存在高水平的N6-甲基腺嘌呤（N6-methyladenosine, m6A）RNA修饰。m6A修饰由METTL3/14复合物催化，该复合物可通过RNA结合蛋白RBM15/15B靶向包括Xist在内的特定RNA分子。已有研究证实，Xist RNA的m6A修饰对于Xist介导的基因沉默过程至关重要。本研究采用CRISPR/Cas9介导的诱变技术，在异种XX型小鼠胚胎干细胞（mouse embryonic stem cells, mESC）中敲除了5'端m6A区域附近的序列。在诱导Xist RNA表达后，我们通过等位基因RNA测序（RNA-seq）检测染色体沉默效果，并通过m6A测序（m6A-seq）分析Xist的m6A修饰模式。此外，我们还借助Xist RNA荧光原位杂交（RNA fluorescence in situ hybridization, RNA FISH）技术，分析了敲除5'端m6A区域对内源性Xist启动子功能的影响。通过纯化表位标记的RBM15蛋白，我们利用质谱/质谱联用（MS/MS）分析技术鉴定了小鼠胚胎干细胞中RBM15的相互作用组。研究结果表明，完整敲除Xist 5'端m6A区域会导致Xist介导的基因沉默出现小幅减弱，且该5'端m6A区域与内源性Xist启动子激活所需的核心DNA元件存在重叠。敲除RBM15结合的Xist A重复序列，可完全阻断Xist 5'端m6A区域的m6A修饰，但不会影响外显子VII区域的修饰水平。我们证实，在小鼠胚胎干细胞中，RBM15可与m6A修饰复合物、SETD1B组蛋白修饰复合物，以及多种参与RNA代谢的蛋白质发生相互作用。

搜集汇总

背景与挑战

背景概述

该数据集通过CRISPR/Cas9基因编辑和多组学技术（RNA-seq、m6A-seq、RNA FISH、质谱分析），研究了Xist RNA 5' m6A修饰区域对X染色体失活的影响，以及RBM15蛋白的相互作用网络。结果表明，5' m6A区域对Xist介导的沉默具有适度调控作用，并包含激活Xist内源启动子的关键DNA元件；RBM15则与m6A修饰复合物及组蛋白修饰复合物相互作用，参与调控过程。