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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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Speckle localization through the RD/RE-rich domain is important in NSrp70-mediated alternative splicing. (A) Schematic diagram of deletion mutants of NSrp70 (left). HEK293T cells were transfected with the indicated construct (1 µg). After 24 h of transfection, cells were visualized in the live chamber by using a confocal microscope equipped with a 100× objective. (B) HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated construct (1 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left top). The ratio of exclusion of Tra2β1 minigene was shown as a histogram (left bottom). GAPDH and β-actin are shown as loading controls. The protein levels of GFP and GFP_NSrp70 mutants were confirmed by western blotting (bottom). Experiment was repeated at least three times to confirm the reproducibility of data.
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Figure 6: Speckle localization through the RD/RE-rich domain is important in NSrp70-mediated alternative splicing. (A) Schematic diagram of deletion mutants of NSrp70 (left). HEK293T cells were transfected with the indicated construct (1 µg). After 24 h of transfection, cells were visualized in the live chamber by using a confocal microscope equipped with a 100× objective. (B) HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated construct (1 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left top). The ratio of exclusion of Tra2β1 minigene was shown as a histogram (left bottom). GAPDH and β-actin are shown as loading controls. The protein levels of GFP and GFP_NSrp70 mutants were confirmed by western blotting (bottom). Experiment was repeated at least three times to confirm the reproducibility of data.

Mentions: Our results thus far demonstrated the requirement for both the N-terminal coiled-coil domain and C-terminal NLS for proper activity of NSrp70, but did not establish the function of the RS-like region through which SC35 and ASF/SF2 are associated. We therefore examined whether the RS-like region is also involved in NSrp70-mediated splicing function. Interestingly, we found that partial deletion of this region (M15; Δ290–471) caused the protein to completely lose its localization in the nuclear speckles and revealed its diffuse pattern in the nucleus, demonstrating that this region contained the sequence for both speckle localization (Figure 6A) and SC35 and ASF/SF2 association (Figure 5E). To further confirm whether the N-terminal region alone is enough to induce splicing activity, the RS-like region was replaced with the foreign sequence containing an 18 amino acid NLS sequence taken from NUCKS (26). Figure 6A shows a schematic map of mutants and their nuclear localization. Interestingly, all mutants showed a similar pattern as M15, suggesting that the RS-like region is important for speckle localization. Consequently, these mutants showed no activity for Tra2β1 exon v2 exclusion in HEK293T cells (Figure 6B). Taken together, although we did not further determine the exact sequence for speckle localization, the region for SC35 and ASF/SF2 association might be overlapping with that for speckle localization. Therefore, our current result suggests that speckle localization is important for the functional role of NSrp70.Figure 6.


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)

Speckle localization through the RD/RE-rich domain is important in NSrp70-mediated alternative splicing. (A) Schematic diagram of deletion mutants of NSrp70 (left). HEK293T cells were transfected with the indicated construct (1 µg). After 24 h of transfection, cells were visualized in the live chamber by using a confocal microscope equipped with a 100× objective. (B) HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated construct (1 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left top). The ratio of exclusion of Tra2β1 minigene was shown as a histogram (left bottom). GAPDH and β-actin are shown as loading controls. The protein levels of GFP and GFP_NSrp70 mutants were confirmed by western blotting (bottom). Experiment was repeated at least three times to confirm the reproducibility of data.
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Related In: Results  -  Collection

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Figure 6: Speckle localization through the RD/RE-rich domain is important in NSrp70-mediated alternative splicing. (A) Schematic diagram of deletion mutants of NSrp70 (left). HEK293T cells were transfected with the indicated construct (1 µg). After 24 h of transfection, cells were visualized in the live chamber by using a confocal microscope equipped with a 100× objective. (B) HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated construct (1 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left top). The ratio of exclusion of Tra2β1 minigene was shown as a histogram (left bottom). GAPDH and β-actin are shown as loading controls. The protein levels of GFP and GFP_NSrp70 mutants were confirmed by western blotting (bottom). Experiment was repeated at least three times to confirm the reproducibility of data.
Mentions: Our results thus far demonstrated the requirement for both the N-terminal coiled-coil domain and C-terminal NLS for proper activity of NSrp70, but did not establish the function of the RS-like region through which SC35 and ASF/SF2 are associated. We therefore examined whether the RS-like region is also involved in NSrp70-mediated splicing function. Interestingly, we found that partial deletion of this region (M15; Δ290–471) caused the protein to completely lose its localization in the nuclear speckles and revealed its diffuse pattern in the nucleus, demonstrating that this region contained the sequence for both speckle localization (Figure 6A) and SC35 and ASF/SF2 association (Figure 5E). To further confirm whether the N-terminal region alone is enough to induce splicing activity, the RS-like region was replaced with the foreign sequence containing an 18 amino acid NLS sequence taken from NUCKS (26). Figure 6A shows a schematic map of mutants and their nuclear localization. Interestingly, all mutants showed a similar pattern as M15, suggesting that the RS-like region is important for speckle localization. Consequently, these mutants showed no activity for Tra2β1 exon v2 exclusion in HEK293T cells (Figure 6B). Taken together, although we did not further determine the exact sequence for speckle localization, the region for SC35 and ASF/SF2 association might be overlapping with that for speckle localization. Therefore, our current result suggests that speckle localization is important for the functional role of NSrp70.Figure 6.

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