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

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. Overall design: To investigate Xist-mediated chromosomal silencing following the depletion of various RNA m6A modification-related proteins, chromatin-associated RNAs (ChrRNA) were isolated to assess allelic gene expression. The dTAG degron strategy was used to rapidly deplete the proteins of interest, minimizing secondary or indirect effects caused by chronic protein inactivation. ChrRNAs were isolated from cells with an endogenous inducible Xist model (iXist-ChrX), derived from mouse embryonic stem cells. The allelic ratios of X-linked genes and Xist RNA expression levels were systematically and comprehensively compared.