m⁶A 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 N⁶-methyladenosine (m⁶A) 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 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 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 m⁶A. These data reveal a pathway of m⁶A formation and recognition required for XIST-mediated transcriptional repression.
Additional Information
© 2016 Macmillan Publishers Limited. Received 29 February; accepted 29 July 2016. Published online 7 September 2016. We thank members of the Jaffrey and Guttman laboratories for comments and suggestions, A. Olarerin-George for discussions on computational analysis, J. -Y. Kim for discussions on immunostaining and imaging, the members of the Weill Cornell Epigenomics Core for their assistance in high-throughput sequencing, A. North and other staff members of the Bio-Imaging Resource Center at the Rockefeller University for their assistance with 3D-SIM imaging, and S. Mukherjee for input on image analysis. This work was supported by NIH grants R01CA186702 (to S.R.J.); T32CA062948 (B.F.P.); T32GM07616 (C.-K.C.) and an NIH Director's Early Independence Award (DP5OD012190), the Rose Hills Foundation, Edward Mallinckrodt Foundation, Sontag Foundation, Searle Scholars Program, the Pew-Stewart Scholars program, and funds from the California Institute of Technology (M.G.). Contributions: All authors designed the experiments and analysed data. D.P.P., B.F.P. and S.R.J. primarily oversaw iCLIP, biochemistry, and SIM experiments; C.-K.C. and M.G. oversaw experiments on XIST-mediated silencing in mouse ES cells. D.P.P. performed the iCLIP experiments, collected data, performed 3D-SIM imaging, analysed data, and prepared figures. C.-K.C. and C.J. performed XIST mouse ES experiments, collected data, and prepared figures. A.C. made and characterized the female pSM33 ES cell line. D.P.P. and S.R.J. wrote the manuscript and all authors provided input and comments. The authors declare no competing financial interests.Attached Files
Accepted Version - nihms870841.pdf
Supplemental Material - nature19342-s1.pdf
Supplemental Material - nature19342-s2.xlsx
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Additional details
- PMCID
- PMC5509218
- Eprint ID
- 70274
- Resolver ID
- CaltechAUTHORS:20160912-092146480
- R01CA186702
- NIH
- T32CA062948
- NIH
- T32GM07616
- NIH
- DP5OD012190
- NIH
- Rose Hills Foundation
- Edward Mallinckrodt Foundation
- Sontag Foundation
- Searle Scholars Program
- Pew Charitable Trust
- Caltech
- Created
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2016-09-12Created from EPrint's datestamp field
- Updated
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2022-04-19Created from EPrint's last_modified field