Nuclear mRNA Degradation Pathway(s) Are Implicated in Xist Regulation and X Chromosome Inactivation

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Abstract
A critical step in X-chromosome inactivation (XCI), which results in the dosage compensation of X-linked gene expression in mammals, is the coating of the presumptive inactive X chromosome by the large noncoding Xist RNA, which then leads to the recruitment of other factors essential for the heterochromatinisation of the inactive X and its transcriptional silencing. In an approach aimed at identifying genes implicated in the X-inactivation process by comparative transcriptional profiling of female and male mouse gastrula, we identified the Eif1 gene involved in translation initiation and RNA degradation. We show here that female embryonic stem cell lines, silenced by RNA interference for the Eif1 gene, are unable to form Xist RNA domains upon differentiation and fail to undergo X-inactivation. To probe further an effect involving RNA degradation pathways, the inhibition by RNA interference of Rent1, a factor essential for nonsense-mediated decay and Exosc10, a specific nuclear component of the exosome, was analysed and shown to similarly impair Xist upregulation and XCI. In Eif1-, Rent1-, and Exosc10-interfered clones, Xist spliced form(s) are strongly downregulated, while the levels of unspliced form(s) of Xist and the stability of Xist RNA remain comparable to that of the control cell lines. Our data suggests a role for mRNA nuclear degradation pathways in the critical regulation of spliced Xist mRNA levels and the onset of the X-inactivation process. In mammals, each cell of the female contains two X chromosomes and hence, potentially a double dose of all X-linked genes when compared to XY males, who carry a single X chromosome. X-inactivation is the mechanism that ensures the dosage-compensation of X-linked gene products between the two sexes. X-inactivation is under the control of a specific region of the X chromosome, the X inactivation center (Xic), which contains the Xist gene encoding a large noncoding RNA transcript whose upregulation is critical to the initiation of X-inactivation. Such changes in steady-state transcript level could be due to altered rates of transcription or changes in the stability and processing of the transcript. How expression of Xist RNA is regulated and the nature of the mechanisms, which lead to Xist upregulation, remain unanswered or only partially answered questions of major importance to the field. In the following article, the authors identify three genes, Eif1, Rent1, and Exosc10, involved in nuclear mRNA degradation pathway(s), which are required for Xist expression upregulation and associated X-inactivation. Inhibition of the function of one or other of these genes leads to a failure of the female cells to undergo X inactivation, suggesting that post-transcriptional nuclear mRNA degradation pathway(s) are essential for the regulation of Xist RNA metabolism and X chromosome inactivation process.