title: A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing.
abstract: 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.
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Benoit
authors.initials: B
authors.lastname: Moindrot
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cerase
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Heather
authors.initials: H
authors.lastname: Coker
authors.affiliation: Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Masui
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Grijzenhout
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Greta
authors.initials: G
authors.lastname: Pintacuda
authors.affiliation: Advanced Cellular Imaging, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Lothar
authors.initials: L
authors.lastname: Schermelleh
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Tatyana B
authors.initials: TB
authors.lastname: Nesterova
authors.affiliation: Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. Electronic address: neil.brockdorff@bioch.ox.ac.uk.
authors.forename: Neil
authors.initials: N
authors.lastname: Brockdorff
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D020868:Gene Silencing; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000072260:RNA Splicing Factors; D062085:RNA, Long Noncoding / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; C519408:Rbm15 protein, mouse; C121253:Spen protein, mouse; C486008:Wtap protein, mouse; C077885:XIST non-coding RNA