N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [ChrRNA-seq; METTL3i]

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 treated the cells (Dox inducible Xist) either with DMSO or METTL3 inhibitor (STM2457) for 6 hours followed by dox-induced Xist expression of 24 hours with continued DMSO/METTL3i treatment. The concentration of METTL3 inhibitor used in this study is 10uM, 20uM, and 30uM. Gene silencing is measured as allelic ratio, i.e. Xi/(Xi+Xa).