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Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

Yamada N, Hasegawa Y, Yue M, Hamada T, Nakagawa S, Ogawa Y - PLoS Genet. (2015)

Bottom Line: Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells.Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells.Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

No MeSH data available.


Related in: MedlinePlus

The creation of the Xist/Tsix double truncation XistdelE7TsixTST6 and Tsix truncation TsixTST6 mutant female ES cells.(A) A map of the Xist/Tsix locus. The positions of the primer pairs used for RT-PCR are indicated with asterisks. SA, splice acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; bpA, beta-actin polyadenylation signal; tpA, tandem polyadenylation signal. (B) 129 and Cast allele-specific RT-qPCR analysis for Tsix RNA at positions T1 and T2, as shown Fig 1A, in TsixTST6 and two XistdelE7TsixTST6 undifferentiated ES cells (#1 and #2). Each value was normalized to that of the wild-type cells (WT) (set to 1), and Gapdh was used as an internal control. Values are given as the mean ± standard deviation (SD) of three independent experiments. (C and D) 129 allele-specific RT-qPCR analysis of the Xist expression at exons 7 and exons 1–3, respectively, was conducted. The expression values were normalized to those of WT at day 0 (set to 1) and Gapdh. The mean ± SD from three independent experiments is shown. (E) Representative allele-specific RT-PCRs for Xist. (F) Quantitative analysis of allele-specific RT-PCRs from three independent experiments including Fig 1E (mean ± SD). (G) Half-life assay for Xist RNA in undifferentiated ES cells and differentiated EBs on day8 upon differentiation. The mean ± SD values from two independent experiments are shown.
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pgen.1005430.g001: The creation of the Xist/Tsix double truncation XistdelE7TsixTST6 and Tsix truncation TsixTST6 mutant female ES cells.(A) A map of the Xist/Tsix locus. The positions of the primer pairs used for RT-PCR are indicated with asterisks. SA, splice acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; bpA, beta-actin polyadenylation signal; tpA, tandem polyadenylation signal. (B) 129 and Cast allele-specific RT-qPCR analysis for Tsix RNA at positions T1 and T2, as shown Fig 1A, in TsixTST6 and two XistdelE7TsixTST6 undifferentiated ES cells (#1 and #2). Each value was normalized to that of the wild-type cells (WT) (set to 1), and Gapdh was used as an internal control. Values are given as the mean ± standard deviation (SD) of three independent experiments. (C and D) 129 allele-specific RT-qPCR analysis of the Xist expression at exons 7 and exons 1–3, respectively, was conducted. The expression values were normalized to those of WT at day 0 (set to 1) and Gapdh. The mean ± SD from three independent experiments is shown. (E) Representative allele-specific RT-PCRs for Xist. (F) Quantitative analysis of allele-specific RT-PCRs from three independent experiments including Fig 1E (mean ± SD). (G) Half-life assay for Xist RNA in undifferentiated ES cells and differentiated EBs on day8 upon differentiation. The mean ± SD values from two independent experiments are shown.

Mentions: To investigate the role of exon 7 in mouse Xist RNA, we inserted two tandem polyadenylation signals (2xtpA) into the end of exon 6 of Xist coupled with splice acceptor (SA)-internal ribosomal entry site (Ires)-hygromycin (Hyg)-pA for Tsix truncation to induce non-random X-inactivation of the mutant X. This resulted in the simultaneous truncation of both Xist and Tsix (XistdelEx7TsixTST6) (Fig 1A and S1 Fig). We used the 16.7 mouse female ES cell line carrying one Mus musculus 129SvJ (129) X-chromosome and one Mus castaneous (Cast) X, which enabled us to perform allele-specific analysis based on single nucleotide polymorphisms (SNPs) between these mouse strains [47,48]. As a control, we also established a Tsix truncation mutant by inserting SA-Ires-Hyg-pA at Xist intron 6, resulting in TsixTST6 female ES cells (Fig 1A and S1 Fig). Allele-specific quantitative RT-PCR (RT-qPCR) analysis of the Tsix expression revealed that the 129 X-chromosome was targeted by the truncation mutation (Fig 1B). While the Tsix expression upstream of the truncation site could be detected by using both the 129 and Cast allele-specific primer sets (T1) in control TsixTST6 and two XistdelEx7/TsixTST6 mutant cells, the Tsix transcript was efficiently truncated downstream (T2, Tsix129 exon 4) of the pA insertion site. These mutant ES cells allow us to address the effect of the Xist mutation on X-inactivation because the Tsix mutation caused a non-random inactivation of the mutant 129 X-chromosome [47].


Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome.

Yamada N, Hasegawa Y, Yue M, Hamada T, Nakagawa S, Ogawa Y - PLoS Genet. (2015)

The creation of the Xist/Tsix double truncation XistdelE7TsixTST6 and Tsix truncation TsixTST6 mutant female ES cells.(A) A map of the Xist/Tsix locus. The positions of the primer pairs used for RT-PCR are indicated with asterisks. SA, splice acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; bpA, beta-actin polyadenylation signal; tpA, tandem polyadenylation signal. (B) 129 and Cast allele-specific RT-qPCR analysis for Tsix RNA at positions T1 and T2, as shown Fig 1A, in TsixTST6 and two XistdelE7TsixTST6 undifferentiated ES cells (#1 and #2). Each value was normalized to that of the wild-type cells (WT) (set to 1), and Gapdh was used as an internal control. Values are given as the mean ± standard deviation (SD) of three independent experiments. (C and D) 129 allele-specific RT-qPCR analysis of the Xist expression at exons 7 and exons 1–3, respectively, was conducted. The expression values were normalized to those of WT at day 0 (set to 1) and Gapdh. The mean ± SD from three independent experiments is shown. (E) Representative allele-specific RT-PCRs for Xist. (F) Quantitative analysis of allele-specific RT-PCRs from three independent experiments including Fig 1E (mean ± SD). (G) Half-life assay for Xist RNA in undifferentiated ES cells and differentiated EBs on day8 upon differentiation. The mean ± SD values from two independent experiments are shown.
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pgen.1005430.g001: The creation of the Xist/Tsix double truncation XistdelE7TsixTST6 and Tsix truncation TsixTST6 mutant female ES cells.(A) A map of the Xist/Tsix locus. The positions of the primer pairs used for RT-PCR are indicated with asterisks. SA, splice acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; bpA, beta-actin polyadenylation signal; tpA, tandem polyadenylation signal. (B) 129 and Cast allele-specific RT-qPCR analysis for Tsix RNA at positions T1 and T2, as shown Fig 1A, in TsixTST6 and two XistdelE7TsixTST6 undifferentiated ES cells (#1 and #2). Each value was normalized to that of the wild-type cells (WT) (set to 1), and Gapdh was used as an internal control. Values are given as the mean ± standard deviation (SD) of three independent experiments. (C and D) 129 allele-specific RT-qPCR analysis of the Xist expression at exons 7 and exons 1–3, respectively, was conducted. The expression values were normalized to those of WT at day 0 (set to 1) and Gapdh. The mean ± SD from three independent experiments is shown. (E) Representative allele-specific RT-PCRs for Xist. (F) Quantitative analysis of allele-specific RT-PCRs from three independent experiments including Fig 1E (mean ± SD). (G) Half-life assay for Xist RNA in undifferentiated ES cells and differentiated EBs on day8 upon differentiation. The mean ± SD values from two independent experiments are shown.
Mentions: To investigate the role of exon 7 in mouse Xist RNA, we inserted two tandem polyadenylation signals (2xtpA) into the end of exon 6 of Xist coupled with splice acceptor (SA)-internal ribosomal entry site (Ires)-hygromycin (Hyg)-pA for Tsix truncation to induce non-random X-inactivation of the mutant X. This resulted in the simultaneous truncation of both Xist and Tsix (XistdelEx7TsixTST6) (Fig 1A and S1 Fig). We used the 16.7 mouse female ES cell line carrying one Mus musculus 129SvJ (129) X-chromosome and one Mus castaneous (Cast) X, which enabled us to perform allele-specific analysis based on single nucleotide polymorphisms (SNPs) between these mouse strains [47,48]. As a control, we also established a Tsix truncation mutant by inserting SA-Ires-Hyg-pA at Xist intron 6, resulting in TsixTST6 female ES cells (Fig 1A and S1 Fig). Allele-specific quantitative RT-PCR (RT-qPCR) analysis of the Tsix expression revealed that the 129 X-chromosome was targeted by the truncation mutation (Fig 1B). While the Tsix expression upstream of the truncation site could be detected by using both the 129 and Cast allele-specific primer sets (T1) in control TsixTST6 and two XistdelEx7/TsixTST6 mutant cells, the Tsix transcript was efficiently truncated downstream (T2, Tsix129 exon 4) of the pA insertion site. These mutant ES cells allow us to address the effect of the Xist mutation on X-inactivation because the Tsix mutation caused a non-random inactivation of the mutant 129 X-chromosome [47].

Bottom Line: Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells.Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells.Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

ABSTRACT
To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.

No MeSH data available.


Related in: MedlinePlus