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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 interacts with ASF/SF2 and SC35 through the RS-like region of NSrp70. (A) HEK293T cells were cotransfected with pEGFP/NSrp70, pmCherry/NSrp70, pmCherry/SC35, pEGFP/ASF/SF2, pEGFP/PML3, pEGFP/SP100 or pEGFP/Daxx. Each transfectant was visualized in the live chamber by using a confocal microscope equipped with a 100× objective. For staining of the endogenous acetylated histone (Act-His), pEGFP/NSrp70-transfected cells were cultured on an 18-mm cover glass for 24 h, and then stained with rabbit polyconal anti-human acetyl-histone H2A (Lys5) antibody followed by TRITC-conjugated anti-rabbit antibody. (B) Coimmunoprecipitation of NSrp70 with ASF/SF2 or SC35. Left: HEK293T cells were transfected with pCS4-3Myc/NSrp70 and the indicated constructs, and then the cell extracts (1 mg) were immunoprecipitated with 50 µl of anti-GFP antibody-conjugated Sepharose 4B slush. Immune complexes were resolved on an SDS–PAGE gel and blotted. The presence of NSrp70 was determined by using an anti-Myc antibody. The expression of indicated constructs was shown by western blotting. Right: HEK293T cells were immunoprecipitated with anti-NSrp70 antibody-conjugated Sepharose 4B slush. Immune complexes were resolved as described above and blotted with an anti-ASF/SF2 or SC35 antibodies. (C–E) HEK293T cells were transfected with pCS4-3Myc/SC35 or pCS4-3Myc/ASF/SF2 and the indicated mutant constructs of NSrp70 (M11, M12, M13, or M15). Immunoprecipitation was performed as described above.
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Figure 5: NSrp70 interacts with ASF/SF2 and SC35 through the RS-like region of NSrp70. (A) HEK293T cells were cotransfected with pEGFP/NSrp70, pmCherry/NSrp70, pmCherry/SC35, pEGFP/ASF/SF2, pEGFP/PML3, pEGFP/SP100 or pEGFP/Daxx. Each transfectant was visualized in the live chamber by using a confocal microscope equipped with a 100× objective. For staining of the endogenous acetylated histone (Act-His), pEGFP/NSrp70-transfected cells were cultured on an 18-mm cover glass for 24 h, and then stained with rabbit polyconal anti-human acetyl-histone H2A (Lys5) antibody followed by TRITC-conjugated anti-rabbit antibody. (B) Coimmunoprecipitation of NSrp70 with ASF/SF2 or SC35. Left: HEK293T cells were transfected with pCS4-3Myc/NSrp70 and the indicated constructs, and then the cell extracts (1 mg) were immunoprecipitated with 50 µl of anti-GFP antibody-conjugated Sepharose 4B slush. Immune complexes were resolved on an SDS–PAGE gel and blotted. The presence of NSrp70 was determined by using an anti-Myc antibody. The expression of indicated constructs was shown by western blotting. Right: HEK293T cells were immunoprecipitated with anti-NSrp70 antibody-conjugated Sepharose 4B slush. Immune complexes were resolved as described above and blotted with an anti-ASF/SF2 or SC35 antibodies. (C–E) HEK293T cells were transfected with pCS4-3Myc/SC35 or pCS4-3Myc/ASF/SF2 and the indicated mutant constructs of NSrp70 (M11, M12, M13, or M15). Immunoprecipitation was performed as described above.

Mentions: As we found that NSrp70 colocalized with SC35, a spliceosomal component (Supplementary Figure S2B), we extended the experiment to test whether NSrp70 is colocalized with other spliceosomal proteins such as ASF/SF2 or non-spliceosomal proteins such as acetylated-histone3, PML3, Sp100 and Daxx. We found that NSrp70 was significantly overlapped with SC35 and ASF/SF2 but not with acetylated-histone3, PML3, Sp100 and Daxx, strongly suggesting that NSrp70 is a new spliceosomal protein (Figure 5A). To prove the physical interactions between NSrp70 and ASF/SF2 or SC35, we generated Myc-tagged NSrp70 and GFP-tagged ASF/SF2 or SC35 and then cotransfected them into HEK293T cells. As shown in Figure 5B (left), immunoprecipitation results revealed that NSrp70 could interact with both ASF/SF2 and SC35 but not with PML3. To test whether native proteins are also physically interacted, endogenous NSrp70 was immunoprecipitated with anti-NSrp70 antibody, and then immunoblotted with anti-ASF/SF2 or anti-SC35 antibodies. As shown in Figure 5B (right), endogenous NSrp70 were also physically interacted with native forms of ASF/SF2 and SC35.Figure 5.


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 interacts with ASF/SF2 and SC35 through the RS-like region of NSrp70. (A) HEK293T cells were cotransfected with pEGFP/NSrp70, pmCherry/NSrp70, pmCherry/SC35, pEGFP/ASF/SF2, pEGFP/PML3, pEGFP/SP100 or pEGFP/Daxx. Each transfectant was visualized in the live chamber by using a confocal microscope equipped with a 100× objective. For staining of the endogenous acetylated histone (Act-His), pEGFP/NSrp70-transfected cells were cultured on an 18-mm cover glass for 24 h, and then stained with rabbit polyconal anti-human acetyl-histone H2A (Lys5) antibody followed by TRITC-conjugated anti-rabbit antibody. (B) Coimmunoprecipitation of NSrp70 with ASF/SF2 or SC35. Left: HEK293T cells were transfected with pCS4-3Myc/NSrp70 and the indicated constructs, and then the cell extracts (1 mg) were immunoprecipitated with 50 µl of anti-GFP antibody-conjugated Sepharose 4B slush. Immune complexes were resolved on an SDS–PAGE gel and blotted. The presence of NSrp70 was determined by using an anti-Myc antibody. The expression of indicated constructs was shown by western blotting. Right: HEK293T cells were immunoprecipitated with anti-NSrp70 antibody-conjugated Sepharose 4B slush. Immune complexes were resolved as described above and blotted with an anti-ASF/SF2 or SC35 antibodies. (C–E) HEK293T cells were transfected with pCS4-3Myc/SC35 or pCS4-3Myc/ASF/SF2 and the indicated mutant constructs of NSrp70 (M11, M12, M13, or M15). Immunoprecipitation was performed as described above.
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Figure 5: NSrp70 interacts with ASF/SF2 and SC35 through the RS-like region of NSrp70. (A) HEK293T cells were cotransfected with pEGFP/NSrp70, pmCherry/NSrp70, pmCherry/SC35, pEGFP/ASF/SF2, pEGFP/PML3, pEGFP/SP100 or pEGFP/Daxx. Each transfectant was visualized in the live chamber by using a confocal microscope equipped with a 100× objective. For staining of the endogenous acetylated histone (Act-His), pEGFP/NSrp70-transfected cells were cultured on an 18-mm cover glass for 24 h, and then stained with rabbit polyconal anti-human acetyl-histone H2A (Lys5) antibody followed by TRITC-conjugated anti-rabbit antibody. (B) Coimmunoprecipitation of NSrp70 with ASF/SF2 or SC35. Left: HEK293T cells were transfected with pCS4-3Myc/NSrp70 and the indicated constructs, and then the cell extracts (1 mg) were immunoprecipitated with 50 µl of anti-GFP antibody-conjugated Sepharose 4B slush. Immune complexes were resolved on an SDS–PAGE gel and blotted. The presence of NSrp70 was determined by using an anti-Myc antibody. The expression of indicated constructs was shown by western blotting. Right: HEK293T cells were immunoprecipitated with anti-NSrp70 antibody-conjugated Sepharose 4B slush. Immune complexes were resolved as described above and blotted with an anti-ASF/SF2 or SC35 antibodies. (C–E) HEK293T cells were transfected with pCS4-3Myc/SC35 or pCS4-3Myc/ASF/SF2 and the indicated mutant constructs of NSrp70 (M11, M12, M13, or M15). Immunoprecipitation was performed as described above.
Mentions: As we found that NSrp70 colocalized with SC35, a spliceosomal component (Supplementary Figure S2B), we extended the experiment to test whether NSrp70 is colocalized with other spliceosomal proteins such as ASF/SF2 or non-spliceosomal proteins such as acetylated-histone3, PML3, Sp100 and Daxx. We found that NSrp70 was significantly overlapped with SC35 and ASF/SF2 but not with acetylated-histone3, PML3, Sp100 and Daxx, strongly suggesting that NSrp70 is a new spliceosomal protein (Figure 5A). To prove the physical interactions between NSrp70 and ASF/SF2 or SC35, we generated Myc-tagged NSrp70 and GFP-tagged ASF/SF2 or SC35 and then cotransfected them into HEK293T cells. As shown in Figure 5B (left), immunoprecipitation results revealed that NSrp70 could interact with both ASF/SF2 and SC35 but not with PML3. To test whether native proteins are also physically interacted, endogenous NSrp70 was immunoprecipitated with anti-NSrp70 antibody, and then immunoblotted with anti-ASF/SF2 or anti-SC35 antibodies. As shown in Figure 5B (right), endogenous NSrp70 were also physically interacted with native forms of ASF/SF2 and SC35.Figure 5.

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