Cell Reports (Jul 2015)

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

  • Benoit Moindrot,
  • Andrea Cerase,
  • Heather Coker,
  • Osamu Masui,
  • Anne Grijzenhout,
  • Greta Pintacuda,
  • Lothar Schermelleh,
  • Tatyana B. Nesterova,
  • Neil Brockdorff

DOI
https://doi.org/10.1016/j.celrep.2015.06.053
Journal volume & issue
Vol. 12, no. 4
pp. 562 – 572

Abstract

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X-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.