m ⁶A RNA methylation promotes XIST-mediated transcriptional repression

Abstract

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