m(6)A RNA methylation promotes XIST-mediated transcriptional repression

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N⁶-methyladenosine (m⁶A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m⁶A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m⁶A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m⁶A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m⁶A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m⁶A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m⁶A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m⁶A. These data reveal a pathway of m⁶A formation and recognition required for XIST-mediated transcriptional repression.