N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [ChrRNA-seq_XistDelm6A]

X chromosome inactivation (XCI) in mammals is orchestrated by the non-coding RNA Xist which together with specific interacting proteins, functions in cis to silence an entire X chromosome. Defined sites on Xist RNA carry the N6-methyladenosine (m6A) modification, and perturbation of the m6A writer complex has been found to abrogate Xist-mediated gene-silencing. However, the relative contribution of m6A and its mechanism of action remain unclear. Here we re-investigate the role of m6A in XCI by applying rapid degron-mediated depletion of METTL3, the catalytic subunit of the m6A writer complex, an approach that minimises indirect effects due to transcriptome-wide depletion of m6A. In contrast to prior studies, we find that acute loss of METTL3/m6A accelerates Xist-mediated gene silencing, and that this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting (NEXT) complex but is independent of the major nuclear m6A reader protein YTHDC1. Our findings demonstrate that the primary function of m6A on Xist RNA is to promote Xist RNA turnover which in turn regulates XCI dynamics. To investigate the role of m⁶A on Xist RNA, we generated deletions in the m⁶A-modified regions of Xist: either a 355 bp deletion in the exon 1 m⁶A region, a 153 bp deletion in the exon 7 m⁶A region, or a combination of both. Cells were either left untreated or induced with doxycycline (dox) for 24 hours to express Xist. Gene silencing was assessed by measuring the allelic ratio, defined as Xi / (Xi + Xa).

哺乳动物X染色体失活（X chromosome inactivation, XCI）由非编码RNA Xist介导调控，其可与特定互作蛋白协同，以顺式作用方式沉默整条X染色体。Xist RNA上的特定位点带有N6-甲基腺嘌呤（N6-methyladenosine, m6A）修饰，且已有研究证实，干扰m6A写入复合物会消除Xist介导的基因沉默效应。然而，m6A在该过程中的相对贡献及其作用机制仍不明朗。本研究通过快速降解子介导的方式耗竭m6A写入复合物的催化亚基METTL3，以最小化因全转录组m6A耗竭带来的间接效应，重新探究m6A在XCI中的作用。与既往研究结果相反，本研究发现METTL3/m6A的急性缺失会加速Xist介导的基因沉默，且该现象与Xist转录本的水平及稳定性提升相关。本研究证实，Xist RNA的降解更新由核外泌体靶向复合物（nuclear exosome targeting, NEXT）介导，但不依赖于主要的核内m6A识别蛋白YTHDC1。本研究结果表明，Xist RNA上m6A修饰的核心功能是促进Xist RNA的降解更新，进而调控X染色体失活的动态过程。为探究m6A在Xist RNA上的作用，本研究对Xist的m6A修饰区域进行了敲除：分别在外显子1的m6A区域进行355 bp缺失、在外显子7的m6A区域进行153 bp缺失，或同时进行两种缺失。实验细胞分为两组：未处理组，以及用多西环素（doxycycline, dox）诱导24小时以表达Xist的诱导组。基因沉默效应通过测定等位基因比率进行评估，该比率定义为Xi/(Xi+Xa)，其中Xi代表失活X染色体（inactive X chromosome），Xa代表活化X染色体（active X chromosome）。