m6A 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 N-6-methyladenosine (m(6)A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m(6)A 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 m(6)A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m(6)A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m(6)A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m(6)A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m(6)A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m(6)A. These data reveal a pathway of m(6)A formation and recognition required for XIST-mediated transcriptional repression.