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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

hnRNP U interacts directly with the first and last exons of mouse and human Xist RNA.(A) UV-crosslinking RIP analysis for Xist RNA and hnRNP U in mouse Neuro2A cells. A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of the primer pairs are shown as arrowheads. The Xist-specific repeats (A-F) defined by Brockdorff et al. [51] are shown. (B) A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of primer pairs are shown as arrowheads. The mean ± SD from four independent experiments is shown.
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pgen.1005430.g004: hnRNP U interacts directly with the first and last exons of mouse and human Xist RNA.(A) UV-crosslinking RIP analysis for Xist RNA and hnRNP U in mouse Neuro2A cells. A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of the primer pairs are shown as arrowheads. The Xist-specific repeats (A-F) defined by Brockdorff et al. [51] are shown. (B) A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of primer pairs are shown as arrowheads. The mean ± SD from four independent experiments is shown.

Mentions: We previously reported that the nuclear matrix protein hnRNP U directly interacts with the Xist RNA via the RGG RNA-binding domain and is involved in the chromosomal localization of Xist RNA along the entire Xi region [35]. To further investigate the interaction between the Xist RNA and the hnRNP U protein across the Xist gene in detail, especially in exon 7 of Xist RNA, UV-crosslinking RNA-immunoprecipitation (UV-crosslinking RIP) analysis was carried out using Neuro2a cells as previously reported (Fig 4A). Multiple primer pairs were designed for the quantitative analysis to examine the binding affinity of hnRNP U to specific regions of the Xist RNA. The UV-crosslinking RIP showed that exons 1 and 7 of the Xist RNA could be significantly co-immunoprecipitated with the FLAG-hnRNP U, unlike the control 7SK, U2, beta-actin and Gapdh RNA. Within exon 1 of the Xist RNA, the second half of the exon (primer positions 4–7) exhibited preferential binding with hnRNP U over the first half of the exon (primer positions 1–3). Interestingly, the hnRNP U also bound to exon 7 of the Xist RNA with as high affinity at primer positions 13–18, with the exception of the repeat E region at primer positions 10–12. We also explored how hnRNP U interacted with the XIST RNA in human cells. Consistent with the data showing that the knockdown of human hnRNP U by siRNA induces the dissociation of XIST RNA from the Xi [43], we confirmed that the hnRNP U knockdown in human HEK293T cells led to the disperse localization of histone macroH2A, which accumulates on the Xi in an Xist RNA-dependent manner (S4 Fig) [19]. These data suggest that hnRNP U is essential for XIST RNA localization on the Xi in both humans and mice. Similar to the interaction between mouse Xist RNA and hnRNP U, the UV-crosslinking RIP analysis revealed that the human hnRNP U preferentially bound to XIST RNA broadly in the second half of the first exon and the last exon, except for the repeat E region (Fig 4B). Overall, our results suggest that exon 7 of the Xist RNA could contribute to the stable localization of Xist RNA on the Xi through interaction with hnRNP U.


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)

hnRNP U interacts directly with the first and last exons of mouse and human Xist RNA.(A) UV-crosslinking RIP analysis for Xist RNA and hnRNP U in mouse Neuro2A cells. A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of the primer pairs are shown as arrowheads. The Xist-specific repeats (A-F) defined by Brockdorff et al. [51] are shown. (B) A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of primer pairs are shown as arrowheads. The mean ± SD from four independent experiments is shown.
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pgen.1005430.g004: hnRNP U interacts directly with the first and last exons of mouse and human Xist RNA.(A) UV-crosslinking RIP analysis for Xist RNA and hnRNP U in mouse Neuro2A cells. A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of the primer pairs are shown as arrowheads. The Xist-specific repeats (A-F) defined by Brockdorff et al. [51] are shown. (B) A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of primer pairs are shown as arrowheads. The mean ± SD from four independent experiments is shown.
Mentions: We previously reported that the nuclear matrix protein hnRNP U directly interacts with the Xist RNA via the RGG RNA-binding domain and is involved in the chromosomal localization of Xist RNA along the entire Xi region [35]. To further investigate the interaction between the Xist RNA and the hnRNP U protein across the Xist gene in detail, especially in exon 7 of Xist RNA, UV-crosslinking RNA-immunoprecipitation (UV-crosslinking RIP) analysis was carried out using Neuro2a cells as previously reported (Fig 4A). Multiple primer pairs were designed for the quantitative analysis to examine the binding affinity of hnRNP U to specific regions of the Xist RNA. The UV-crosslinking RIP showed that exons 1 and 7 of the Xist RNA could be significantly co-immunoprecipitated with the FLAG-hnRNP U, unlike the control 7SK, U2, beta-actin and Gapdh RNA. Within exon 1 of the Xist RNA, the second half of the exon (primer positions 4–7) exhibited preferential binding with hnRNP U over the first half of the exon (primer positions 1–3). Interestingly, the hnRNP U also bound to exon 7 of the Xist RNA with as high affinity at primer positions 13–18, with the exception of the repeat E region at primer positions 10–12. We also explored how hnRNP U interacted with the XIST RNA in human cells. Consistent with the data showing that the knockdown of human hnRNP U by siRNA induces the dissociation of XIST RNA from the Xi [43], we confirmed that the hnRNP U knockdown in human HEK293T cells led to the disperse localization of histone macroH2A, which accumulates on the Xi in an Xist RNA-dependent manner (S4 Fig) [19]. These data suggest that hnRNP U is essential for XIST RNA localization on the Xi in both humans and mice. Similar to the interaction between mouse Xist RNA and hnRNP U, the UV-crosslinking RIP analysis revealed that the human hnRNP U preferentially bound to XIST RNA broadly in the second half of the first exon and the last exon, except for the repeat E region (Fig 4B). Overall, our results suggest that exon 7 of the Xist RNA could contribute to the stable localization of Xist RNA on the Xi through interaction with hnRNP U.

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