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NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo.

Kim YD, Lee JY, Oh KM, Araki M, Araki K, Yamamura K, Jun CD - Nucleic Acids Res. (2011)

Bottom Line: Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo.The N-terminal region (107-161) was essential for the pre-mRNA splicing activity.Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Cell Dynamics Research Center, and Immune Synapse Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.

ABSTRACT
Nuclear speckles are known to be the storage sites of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its subcellular localization and apparent molecular weight. This protein was first identified as CCDC55 by the National Institutes of Health Mammalian Gene Collection, although its function has not been assigned. NSrp70 was colocalized and physically interacted with SC35 and ASF/SF2 in speckles. NSrp70 has a putative RNA recognition motif, the RS-like region, and two coiled-coil domains, suggesting a role in RNA processing. Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal 10 amino acids (531-540), including (536)RD(537), were identified as a novel nuclear localization signal, and the region spanning 290-471 amino acids was critical for speckle localization and binding to SC35 and ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice led to a lack of progeny, including fetal embryos. Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.

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NSrp70-deficient homozygous mouse exhibits early embryonic lethality. (A) Schematic diagram of the exchangeable Gene-Trap system. Note: numbered boxes, exons; lines, introns; diagonal lines, regions of homology where crossover can take place; β-geo, encoding a fusion protein with both β-galactosidase and neomycin phosphotransferase activity; SA, splicing acceptor; pA, polyadenine. Representative gel data revealed the PCR products of NSrp70+/+ and NSrp70+/GT. Primers used target the region from EX1 to EX2 (1736 bp) and the trap vector (605 bp), respectively. (B) Intercrossing the heterozygous mouse (NSrp70+/GT) produced no offspring homozygous for the allele containing the Gene-Trap vector (GT). The bar graph represents the number of offspring litters from the intercrossed NSrp70+/GT mice. (C) Body weight changes in NSrp70+/+ and NSrp70+/GT mice. Mice were weighed every week through the entire experimental period indicated. Data represent mean ± SEM (n = 8). (D) Western blot analysis was performed using rabbit polyclonal anti-NSrp70 antibody with mouse tissues from NSrp70+/+ and NSrp70+/GT (50 µg for each lane). mGAPDH was used as a loading control. (E) Lymphocytes from NSrp70+/+ and NSrp70+/GT mouse were stimulated with PMA (200 nM) and A23187 (1 µM) for 24 h, and then the expression of mIL-2 and mNSrp70 mRNA was determined by RT–PCR. mGAPDH was used as a loading control. (F) Lymphocytes (5 × 105) from NSrp70+/+ or NSrp70+/GT mice were allowed to migrate to the SDF-α (50 ng/ml)-containing lower well for 2.5 h at 37°C. The bar graph represents quantitative determinations of migrated cells obtained from three different fields of lower compartment.
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Figure 7: NSrp70-deficient homozygous mouse exhibits early embryonic lethality. (A) Schematic diagram of the exchangeable Gene-Trap system. Note: numbered boxes, exons; lines, introns; diagonal lines, regions of homology where crossover can take place; β-geo, encoding a fusion protein with both β-galactosidase and neomycin phosphotransferase activity; SA, splicing acceptor; pA, polyadenine. Representative gel data revealed the PCR products of NSrp70+/+ and NSrp70+/GT. Primers used target the region from EX1 to EX2 (1736 bp) and the trap vector (605 bp), respectively. (B) Intercrossing the heterozygous mouse (NSrp70+/GT) produced no offspring homozygous for the allele containing the Gene-Trap vector (GT). The bar graph represents the number of offspring litters from the intercrossed NSrp70+/GT mice. (C) Body weight changes in NSrp70+/+ and NSrp70+/GT mice. Mice were weighed every week through the entire experimental period indicated. Data represent mean ± SEM (n = 8). (D) Western blot analysis was performed using rabbit polyclonal anti-NSrp70 antibody with mouse tissues from NSrp70+/+ and NSrp70+/GT (50 µg for each lane). mGAPDH was used as a loading control. (E) Lymphocytes from NSrp70+/+ and NSrp70+/GT mouse were stimulated with PMA (200 nM) and A23187 (1 µM) for 24 h, and then the expression of mIL-2 and mNSrp70 mRNA was determined by RT–PCR. mGAPDH was used as a loading control. (F) Lymphocytes (5 × 105) from NSrp70+/+ or NSrp70+/GT mice were allowed to migrate to the SDF-α (50 ng/ml)-containing lower well for 2.5 h at 37°C. The bar graph represents quantitative determinations of migrated cells obtained from three different fields of lower compartment.

Mentions: Because human NSrp70 has high similarity (75.6%) with its mouse ortholog, as depicted in Supplementary Figure S3, we investigated the role of NSrp70 in a mouse system generated in a large-scale Gene-Trap project (27). By sequence analysis of DNA isolated from NSrp70+/GT (Gene-Trap) heterozygous mice, we confirmed that the retroviral Gene-Trap vector was inserted in intron 1 of the mouse NSrp70 gene on chromosome 11, downstream of the exon encoding the initiation methionine (Figure 7A). To generate NSrp70GT/GT mice, heterozygous mutant mice were intercrossed and the genotypes of the offspring were analyzed by PCR. Among more than 95 offspring investigated, only NSrp70+/GT (∼80%) and NSrp70+/+ (∼20%) mice, but not viable NSrp70GT/GT mice, were detected (Fig. 7B), leading to the assumption that knockout of NSrp70 in vivo results in embryonic lethality. To determine in which developmental stage knockout of NSrp70 induces lethality, we carried out genotyping of embryos from embryonic day (E) 6.5 to 19.5. However, no homozygous embryo was observed (data not shown), suggesting that homozygous mutation leads to embryonic lethality shortly after implantation. In contrast, heterozygous mice (NSrp70+/GT) showed no differences to wild-type (NSrp70+/+) with regard to body weight and expression levels of NSrp70 protein (Figure 7C and D). In addition, no differences were observed in terms of IL-2 response in splenocytes, as evaluated by mitogen stimulation and migration of isolated T cells by SDF-1α (Figure 7E and F), suggesting that NSrp70 has a recessive phenotype in mice.Figure 7.


NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo.

Kim YD, Lee JY, Oh KM, Araki M, Araki K, Yamamura K, Jun CD - Nucleic Acids Res. (2011)

NSrp70-deficient homozygous mouse exhibits early embryonic lethality. (A) Schematic diagram of the exchangeable Gene-Trap system. Note: numbered boxes, exons; lines, introns; diagonal lines, regions of homology where crossover can take place; β-geo, encoding a fusion protein with both β-galactosidase and neomycin phosphotransferase activity; SA, splicing acceptor; pA, polyadenine. Representative gel data revealed the PCR products of NSrp70+/+ and NSrp70+/GT. Primers used target the region from EX1 to EX2 (1736 bp) and the trap vector (605 bp), respectively. (B) Intercrossing the heterozygous mouse (NSrp70+/GT) produced no offspring homozygous for the allele containing the Gene-Trap vector (GT). The bar graph represents the number of offspring litters from the intercrossed NSrp70+/GT mice. (C) Body weight changes in NSrp70+/+ and NSrp70+/GT mice. Mice were weighed every week through the entire experimental period indicated. Data represent mean ± SEM (n = 8). (D) Western blot analysis was performed using rabbit polyclonal anti-NSrp70 antibody with mouse tissues from NSrp70+/+ and NSrp70+/GT (50 µg for each lane). mGAPDH was used as a loading control. (E) Lymphocytes from NSrp70+/+ and NSrp70+/GT mouse were stimulated with PMA (200 nM) and A23187 (1 µM) for 24 h, and then the expression of mIL-2 and mNSrp70 mRNA was determined by RT–PCR. mGAPDH was used as a loading control. (F) Lymphocytes (5 × 105) from NSrp70+/+ or NSrp70+/GT mice were allowed to migrate to the SDF-α (50 ng/ml)-containing lower well for 2.5 h at 37°C. The bar graph represents quantitative determinations of migrated cells obtained from three different fields of lower compartment.
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Figure 7: NSrp70-deficient homozygous mouse exhibits early embryonic lethality. (A) Schematic diagram of the exchangeable Gene-Trap system. Note: numbered boxes, exons; lines, introns; diagonal lines, regions of homology where crossover can take place; β-geo, encoding a fusion protein with both β-galactosidase and neomycin phosphotransferase activity; SA, splicing acceptor; pA, polyadenine. Representative gel data revealed the PCR products of NSrp70+/+ and NSrp70+/GT. Primers used target the region from EX1 to EX2 (1736 bp) and the trap vector (605 bp), respectively. (B) Intercrossing the heterozygous mouse (NSrp70+/GT) produced no offspring homozygous for the allele containing the Gene-Trap vector (GT). The bar graph represents the number of offspring litters from the intercrossed NSrp70+/GT mice. (C) Body weight changes in NSrp70+/+ and NSrp70+/GT mice. Mice were weighed every week through the entire experimental period indicated. Data represent mean ± SEM (n = 8). (D) Western blot analysis was performed using rabbit polyclonal anti-NSrp70 antibody with mouse tissues from NSrp70+/+ and NSrp70+/GT (50 µg for each lane). mGAPDH was used as a loading control. (E) Lymphocytes from NSrp70+/+ and NSrp70+/GT mouse were stimulated with PMA (200 nM) and A23187 (1 µM) for 24 h, and then the expression of mIL-2 and mNSrp70 mRNA was determined by RT–PCR. mGAPDH was used as a loading control. (F) Lymphocytes (5 × 105) from NSrp70+/+ or NSrp70+/GT mice were allowed to migrate to the SDF-α (50 ng/ml)-containing lower well for 2.5 h at 37°C. The bar graph represents quantitative determinations of migrated cells obtained from three different fields of lower compartment.
Mentions: Because human NSrp70 has high similarity (75.6%) with its mouse ortholog, as depicted in Supplementary Figure S3, we investigated the role of NSrp70 in a mouse system generated in a large-scale Gene-Trap project (27). By sequence analysis of DNA isolated from NSrp70+/GT (Gene-Trap) heterozygous mice, we confirmed that the retroviral Gene-Trap vector was inserted in intron 1 of the mouse NSrp70 gene on chromosome 11, downstream of the exon encoding the initiation methionine (Figure 7A). To generate NSrp70GT/GT mice, heterozygous mutant mice were intercrossed and the genotypes of the offspring were analyzed by PCR. Among more than 95 offspring investigated, only NSrp70+/GT (∼80%) and NSrp70+/+ (∼20%) mice, but not viable NSrp70GT/GT mice, were detected (Fig. 7B), leading to the assumption that knockout of NSrp70 in vivo results in embryonic lethality. To determine in which developmental stage knockout of NSrp70 induces lethality, we carried out genotyping of embryos from embryonic day (E) 6.5 to 19.5. However, no homozygous embryo was observed (data not shown), suggesting that homozygous mutation leads to embryonic lethality shortly after implantation. In contrast, heterozygous mice (NSrp70+/GT) showed no differences to wild-type (NSrp70+/+) with regard to body weight and expression levels of NSrp70 protein (Figure 7C and D). In addition, no differences were observed in terms of IL-2 response in splenocytes, as evaluated by mitogen stimulation and migration of isolated T cells by SDF-1α (Figure 7E and F), suggesting that NSrp70 has a recessive phenotype in mice.Figure 7.

Bottom Line: Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo.The N-terminal region (107-161) was essential for the pre-mRNA splicing activity.Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Cell Dynamics Research Center, and Immune Synapse Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.

ABSTRACT
Nuclear speckles are known to be the storage sites of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its subcellular localization and apparent molecular weight. This protein was first identified as CCDC55 by the National Institutes of Health Mammalian Gene Collection, although its function has not been assigned. NSrp70 was colocalized and physically interacted with SC35 and ASF/SF2 in speckles. NSrp70 has a putative RNA recognition motif, the RS-like region, and two coiled-coil domains, suggesting a role in RNA processing. Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal 10 amino acids (531-540), including (536)RD(537), were identified as a novel nuclear localization signal, and the region spanning 290-471 amino acids was critical for speckle localization and binding to SC35 and ASF/SF2. The N-terminal region (107-161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice led to a lack of progeny, including fetal embryos. Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.

Show MeSH
Related in: MedlinePlus