m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics

Abstract

X-chromosome inactivation (XCI) in mammals is orchestrated by the noncoding RNA X-inactive-specific transcript (Xist) that, 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 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 minimizes indirect effects because of transcriptome-wide depletion of m⁶A. We find that acute loss of METTL3 and m⁶A accelerates Xist-mediated gene silencing and this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting complex but is independent of the principal 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.