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RNA（non-coding RNA，ncRNA）Xist，后者可抑制其自身转录所在染色体的基因活性。Xist外显子I（紧邻转录本5'端）以及3'端远端的Xist外显子VII区域内，均存在高水平的N6-甲基腺嘌呤核苷（N6-methyladenosine，m6A）RNA修饰。该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靶向结合包括Xist在内的特定靶标RNA。已有研究证实，Xist RNA的m6A修饰对Xist介导的基因沉默过程具有关键作用。
本研究借助CRISPR/Cas9介导的诱变技术，在种间XX型小鼠胚胎干细胞（mouse embryonic stem cells, mESC）中敲除了Xist 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）检测染色体沉默水平，并利用m6A测序（m6A-seq）分析Xist的m6A修饰模式。此外，我们还采用RNA荧光原位杂交（RNA FISH）技术，探究了敲除5'端m6A区域对内源性Xist启动子功能的影响。
通过纯化带有表位标签的RBM15蛋白，我们运用质谱联用（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：敲除整个Xist 5'端m6A区域会导致Xist介导的基因沉默作用轻度减弱；且该5'端m6A区域与内源性Xist启动子激活所必需的DNA核心元件存在重叠。进一步实验发现，敲除RBM15结合的Xist A重复序列，可完全消除Xist 5'端m6A区域的m6A修饰，但对外显子VII区域的修饰无显著影响。此外，我们在小鼠胚胎干细胞中证实，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代谢的蛋白发生相互作用。