The role of the Xist 5’ m6A region and RBM15 in X chromosome inactivation

Published: 3 Feb 2020 | Version 3 | DOI: 10.17632/56f2m8nd9h.3
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Description of this data

X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5’ end of the transcript, and also further 3’, in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist–mediated gene silencing. We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5’ m6A region in interspecific XX mouse embryonic stem cells (mESC). Following induction of Xist RNA expression we assay chromosome silencing using allelic RNA-seq and Xist m6A patterns using m6A-seq. Additionally we use Xist RNA FISH to analyse the effect of deleting the 5’ m6A region on the function of the endogenous Xist promoter. Purifying epitope tagged RBM15, we employ MS/MS analysis to define the RBM15 interactome in mESCs. We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing, and that the 5’ m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5’ m6A region without affecting the modification in exon VII. We show that in mESCs RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism

Experiment data files

  • Figure 2: Raw RNA-FISH images and scoring data
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  • Figure 4: Raw western blot and gel data (RBM15 purification for mass spec)
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  • Methods: Plasmid sequences
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Latest version

  • Version 3

    2020-02-03

    Published: 2020-02-03

    DOI: 10.17632/56f2m8nd9h.3

    Cite this dataset

    Coker, Heather; Brockdorff, Neil; Wei, Guifeng; Moindrot, Benoit; Nesterova, Tatyana; Mohammed, Shabaz (2020), “The role of the Xist 5’ m6A region and RBM15 in X chromosome inactivation”, Mendeley Data, v3 http://dx.doi.org/10.17632/56f2m8nd9h.3

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Biochemistry, Genetics, Epigenetics

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