N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [SLAM-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. To investigate the stability of Xist RNA following the depletion of various m6A RNA modification-related proteins (METTL3, YTHDC1, ZCCHC8), the dTAG degron system was employed. This method allows for rapid protein depletion, thereby minimizing secondary or indirect effects that may arise from prolonged protein inactivation. The SLAMseq strategy was used to analyze total RNA. Total RNA was isolated from cells of an inducible Xist model (iXist-ChrX) with a Bgl stem loop inserted in exon 7, derived from mouse embryonic stem cells.