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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 exon 7 truncation in Xist RNA impairs the interaction between hnRNP U and Xist RNA.(A) Overview showing the generation of the FLAG-HA knock-in hnRNP U allele. The protein coding or 5´ UTR regions are shown as a gray or white box, respectively. The FLAG-HA tag is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´ UTR regions are capitalized or lowercased, respectively. The sgRNA sequence is labeled in green. The protospacer-adjacent motif (PAM) sequence is labeled in red. (B) PCR genotyping analysis of the FLAG-HA tag knock-in hnRNP U allele. Genomic PCR using primers hnRNPU-utrF and hnRNPU-codeR amplified 212 or 284 bp PCR products from the wild-type or the FLAG-HA knock-in alleles, respectively. All FLAG-HA targeted knock-in ES cells shown in this work are homozygous knock-in clones. (C) Quantitative RT-PCR analysis of FLAGHA-hnRNP U using hnRNPU-F and hnRNPU-R in the parental and targeted ES cell lines at day 12 upon differentiation. The mean ± SEM from three independent experiments is shown. (D) A representative western blotting analysis using anti-FLAG, hnRNP U and tubulin antibodies with the parental and FLAG-HA hnRNP U knock-in ES clones at day 12 upon differentiation. (E) Quantification analysis of the hnRNP U western blotting, including Fig 5D. Each was normalized to the parental TST6 cells (set to 1). The mean ± SD values from three independent experiments are shown. (F) UV-crosslinking RIP analysis using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing FLAG-HA-tagged hnRNP U upon differentiation at day 12. The mean ± SD bar from three independent experiments is shown with an unpaired t test P values (*p<0.05, **p<0.01).
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pgen.1005430.g005: The exon 7 truncation in Xist RNA impairs the interaction between hnRNP U and Xist RNA.(A) Overview showing the generation of the FLAG-HA knock-in hnRNP U allele. The protein coding or 5´ UTR regions are shown as a gray or white box, respectively. The FLAG-HA tag is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´ UTR regions are capitalized or lowercased, respectively. The sgRNA sequence is labeled in green. The protospacer-adjacent motif (PAM) sequence is labeled in red. (B) PCR genotyping analysis of the FLAG-HA tag knock-in hnRNP U allele. Genomic PCR using primers hnRNPU-utrF and hnRNPU-codeR amplified 212 or 284 bp PCR products from the wild-type or the FLAG-HA knock-in alleles, respectively. All FLAG-HA targeted knock-in ES cells shown in this work are homozygous knock-in clones. (C) Quantitative RT-PCR analysis of FLAGHA-hnRNP U using hnRNPU-F and hnRNPU-R in the parental and targeted ES cell lines at day 12 upon differentiation. The mean ± SEM from three independent experiments is shown. (D) A representative western blotting analysis using anti-FLAG, hnRNP U and tubulin antibodies with the parental and FLAG-HA hnRNP U knock-in ES clones at day 12 upon differentiation. (E) Quantification analysis of the hnRNP U western blotting, including Fig 5D. Each was normalized to the parental TST6 cells (set to 1). The mean ± SD values from three independent experiments are shown. (F) UV-crosslinking RIP analysis using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing FLAG-HA-tagged hnRNP U upon differentiation at day 12. The mean ± SD bar from three independent experiments is shown with an unpaired t test P values (*p<0.05, **p<0.01).

Mentions: Next, to investigate whether the unstable Xist RNA localization and X-linked gene silencing on the Xi observed in the XistdelEx7TsixTST6 mutant cells was due to the disturbed interaction between hnRNP U and the Xist RNA lacking exon 7, we performed UV-crosslinked RIP using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing a FLAG-HA epitope tag hnRNP U. To establish the ES cell lines expressing the FLAG-HA tagged hnRNP U from the endogenous hnRNP U loci, we used the CRISPR/Cas system. With this system, we obtained 2 and 3 FLAG-HA biallelic knock-in ES cell lines derived from the TsixTST6 and XistdelEx7TsixTST6 mutant ES cells, respectively (Fig 5A and 5B). By RT-qPCR and western blotting using anti-FLAG and anti-hnRNP U antibodies, we confirmed that the FLAG-HA knock-in ES cell lines expressed similar levels of FLAG-HA-hnRNP U to those of the parental ES cell lines (the TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines) on day 12 upon differentiation (Fig 5C–5E). We also confirmed that the FLAG-HA knock-in at the endogenous hnRNP U did not alter the kinetics of Xist expression upon differentiation and X-linked gene silencing compared to the parental ES cell lines (Figs 1D and 3A, S5A and S5B Fig). Furthermore, upon differentiation on day 12, the TsixTST6 and XistdelEx7TsixTST6 mutant EB cells expressing the FLAG-HA-hnRNP U from the endogenous loci exhibited a similar proportion of Xist RNA- and H3K27me3-positive cells to those observed in their parental cell lines (Fig 2 and S5C Fig).


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 exon 7 truncation in Xist RNA impairs the interaction between hnRNP U and Xist RNA.(A) Overview showing the generation of the FLAG-HA knock-in hnRNP U allele. The protein coding or 5´ UTR regions are shown as a gray or white box, respectively. The FLAG-HA tag is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´ UTR regions are capitalized or lowercased, respectively. The sgRNA sequence is labeled in green. The protospacer-adjacent motif (PAM) sequence is labeled in red. (B) PCR genotyping analysis of the FLAG-HA tag knock-in hnRNP U allele. Genomic PCR using primers hnRNPU-utrF and hnRNPU-codeR amplified 212 or 284 bp PCR products from the wild-type or the FLAG-HA knock-in alleles, respectively. All FLAG-HA targeted knock-in ES cells shown in this work are homozygous knock-in clones. (C) Quantitative RT-PCR analysis of FLAGHA-hnRNP U using hnRNPU-F and hnRNPU-R in the parental and targeted ES cell lines at day 12 upon differentiation. The mean ± SEM from three independent experiments is shown. (D) A representative western blotting analysis using anti-FLAG, hnRNP U and tubulin antibodies with the parental and FLAG-HA hnRNP U knock-in ES clones at day 12 upon differentiation. (E) Quantification analysis of the hnRNP U western blotting, including Fig 5D. Each was normalized to the parental TST6 cells (set to 1). The mean ± SD values from three independent experiments are shown. (F) UV-crosslinking RIP analysis using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing FLAG-HA-tagged hnRNP U upon differentiation at day 12. The mean ± SD bar from three independent experiments is shown with an unpaired t test P values (*p<0.05, **p<0.01).
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pgen.1005430.g005: The exon 7 truncation in Xist RNA impairs the interaction between hnRNP U and Xist RNA.(A) Overview showing the generation of the FLAG-HA knock-in hnRNP U allele. The protein coding or 5´ UTR regions are shown as a gray or white box, respectively. The FLAG-HA tag is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´ UTR regions are capitalized or lowercased, respectively. The sgRNA sequence is labeled in green. The protospacer-adjacent motif (PAM) sequence is labeled in red. (B) PCR genotyping analysis of the FLAG-HA tag knock-in hnRNP U allele. Genomic PCR using primers hnRNPU-utrF and hnRNPU-codeR amplified 212 or 284 bp PCR products from the wild-type or the FLAG-HA knock-in alleles, respectively. All FLAG-HA targeted knock-in ES cells shown in this work are homozygous knock-in clones. (C) Quantitative RT-PCR analysis of FLAGHA-hnRNP U using hnRNPU-F and hnRNPU-R in the parental and targeted ES cell lines at day 12 upon differentiation. The mean ± SEM from three independent experiments is shown. (D) A representative western blotting analysis using anti-FLAG, hnRNP U and tubulin antibodies with the parental and FLAG-HA hnRNP U knock-in ES clones at day 12 upon differentiation. (E) Quantification analysis of the hnRNP U western blotting, including Fig 5D. Each was normalized to the parental TST6 cells (set to 1). The mean ± SD values from three independent experiments are shown. (F) UV-crosslinking RIP analysis using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing FLAG-HA-tagged hnRNP U upon differentiation at day 12. The mean ± SD bar from three independent experiments is shown with an unpaired t test P values (*p<0.05, **p<0.01).
Mentions: Next, to investigate whether the unstable Xist RNA localization and X-linked gene silencing on the Xi observed in the XistdelEx7TsixTST6 mutant cells was due to the disturbed interaction between hnRNP U and the Xist RNA lacking exon 7, we performed UV-crosslinked RIP using TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines expressing a FLAG-HA epitope tag hnRNP U. To establish the ES cell lines expressing the FLAG-HA tagged hnRNP U from the endogenous hnRNP U loci, we used the CRISPR/Cas system. With this system, we obtained 2 and 3 FLAG-HA biallelic knock-in ES cell lines derived from the TsixTST6 and XistdelEx7TsixTST6 mutant ES cells, respectively (Fig 5A and 5B). By RT-qPCR and western blotting using anti-FLAG and anti-hnRNP U antibodies, we confirmed that the FLAG-HA knock-in ES cell lines expressed similar levels of FLAG-HA-hnRNP U to those of the parental ES cell lines (the TsixTST6 and XistdelEx7TsixTST6 mutant ES cell lines) on day 12 upon differentiation (Fig 5C–5E). We also confirmed that the FLAG-HA knock-in at the endogenous hnRNP U did not alter the kinetics of Xist expression upon differentiation and X-linked gene silencing compared to the parental ES cell lines (Figs 1D and 3A, S5A and S5B Fig). Furthermore, upon differentiation on day 12, the TsixTST6 and XistdelEx7TsixTST6 mutant EB cells expressing the FLAG-HA-hnRNP U from the endogenous loci exhibited a similar proportion of Xist RNA- and H3K27me3-positive cells to those observed in their parental cell lines (Fig 2 and S5C Fig).

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