A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing

•An shRNA screen identifies factors implicated in chromosome silencing by Xist RNA•Rbm15, Wtap, and Spen are required for Xist-mediated silencing•Rbm15 is important for efficient deposition of H3K27me3 on the inactive chromosome•Rbm15, Wtap, and Spen co-localize with Xist RNA in perichromatin spaces

SummaryX-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.

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