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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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Nuclear localization is mediated by a novel NLS sequence and is required for NSrp70-mediated alternative splicing. (A–C) Schematic diagram of deletion or substitution mutants of NSrp70 (left) and their subcellular localization in HEK293T cells (right). HEK293T cells were transfected with pEGFP vectors (1 µg) that contain wild-type or mutant NSrp70. After 24 h of transfection, cells were fixed, stained with anti-SC35 antibody followed by TRITC-conjugated anti-mouse antibody, and visualized by confocal microscopy (right). Sequence alignment of the NLS sequence with other species, as analyzed by Clustal X and GeneDoc, is depicted in (C) (left bottom). (D) Nuclear localization is required for NSrp70-mediated exon 2 exclusion of Tra2β1 minigene. HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated NSrp70 constructs (2 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left). The ratio of exclusion or inclusion of Tra2β1 or CD44 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 3: Nuclear localization is mediated by a novel NLS sequence and is required for NSrp70-mediated alternative splicing. (A–C) Schematic diagram of deletion or substitution mutants of NSrp70 (left) and their subcellular localization in HEK293T cells (right). HEK293T cells were transfected with pEGFP vectors (1 µg) that contain wild-type or mutant NSrp70. After 24 h of transfection, cells were fixed, stained with anti-SC35 antibody followed by TRITC-conjugated anti-mouse antibody, and visualized by confocal microscopy (right). Sequence alignment of the NLS sequence with other species, as analyzed by Clustal X and GeneDoc, is depicted in (C) (left bottom). (D) Nuclear localization is required for NSrp70-mediated exon 2 exclusion of Tra2β1 minigene. HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated NSrp70 constructs (2 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left). The ratio of exclusion or inclusion of Tra2β1 or CD44 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: The RS domain of SR or SR-related proteins is thought to be required for protein–protein interaction with each other or with other components of the splicing machineries (5,7,20) as well as for subcellular localization (21,22). As we found an RS-like region in NSrp70, which resembles the RS domain in other SR proteins, we hypothesized that this region may contain the NLS sequence. To this end, we generated a series of GFP-tagged deletion mutants for probing NLS sequence in the putative RS-like region of NSrp70. After transient transfection, the localization of GFP-tagged NSrp70 mutants was analyzed by confocal microscopy. To better monitor the localization, the fixed cells were also counter stained with endogenous SC35. In contrast to the wild-type (GFP_NSrp70), deletion of amino acids spanning the 439–558 (M1) region appeared to be localized in the cytosol, suggesting that this region included the NLS sequence (Figure 3A). To find the exact NLS sequence, we therefore designed more NSrp70 mutant constructs (Figure 3B). Interestingly, we found that mutant M10 (Δ531–540) localized in the cytosol, suggesting that this region belongs to the NLS sequence of NSrp70. Further point mutations at 536R/A or 537D/A demonstrated that these two amino acids are critical residues for nuclear localization and target the speckle compartment (Figure 3C). To extend these findings with regard to the functional role, we performed in vivo splicing assay with Tra2β1 minigene. M1 (Δ439–558), M8 (Δ531–558) and M10 (Δ531–540), which localize in the cytosol, did not induce alternative splicing (Figure 3D). Interestingly, however, M7 (Δ541–558), which localizes in the nucleus, affected the alternative splice site selection and is comparable to that of the wild-type (Figure 3D). These results demonstrate that nuclear localization by the NLS sequence (531–540), including 536RD537, is important for the functional role of NSrp70. Further, we found that this sequence does not match the known NLS sequence, suggesting that this is a novel NLS sequence.Figure 3.


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)

Nuclear localization is mediated by a novel NLS sequence and is required for NSrp70-mediated alternative splicing. (A–C) Schematic diagram of deletion or substitution mutants of NSrp70 (left) and their subcellular localization in HEK293T cells (right). HEK293T cells were transfected with pEGFP vectors (1 µg) that contain wild-type or mutant NSrp70. After 24 h of transfection, cells were fixed, stained with anti-SC35 antibody followed by TRITC-conjugated anti-mouse antibody, and visualized by confocal microscopy (right). Sequence alignment of the NLS sequence with other species, as analyzed by Clustal X and GeneDoc, is depicted in (C) (left bottom). (D) Nuclear localization is required for NSrp70-mediated exon 2 exclusion of Tra2β1 minigene. HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated NSrp70 constructs (2 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left). The ratio of exclusion or inclusion of Tra2β1 or CD44 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 3: Nuclear localization is mediated by a novel NLS sequence and is required for NSrp70-mediated alternative splicing. (A–C) Schematic diagram of deletion or substitution mutants of NSrp70 (left) and their subcellular localization in HEK293T cells (right). HEK293T cells were transfected with pEGFP vectors (1 µg) that contain wild-type or mutant NSrp70. After 24 h of transfection, cells were fixed, stained with anti-SC35 antibody followed by TRITC-conjugated anti-mouse antibody, and visualized by confocal microscopy (right). Sequence alignment of the NLS sequence with other species, as analyzed by Clustal X and GeneDoc, is depicted in (C) (left bottom). (D) Nuclear localization is required for NSrp70-mediated exon 2 exclusion of Tra2β1 minigene. HEK293T cells were cotransfected with Tra2β1 minigene (2 µg) and the indicated NSrp70 constructs (2 µg). After 24 h of transfection, exon v2 exclusion was determined by RT–PCR (left). The ratio of exclusion or inclusion of Tra2β1 or CD44 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: The RS domain of SR or SR-related proteins is thought to be required for protein–protein interaction with each other or with other components of the splicing machineries (5,7,20) as well as for subcellular localization (21,22). As we found an RS-like region in NSrp70, which resembles the RS domain in other SR proteins, we hypothesized that this region may contain the NLS sequence. To this end, we generated a series of GFP-tagged deletion mutants for probing NLS sequence in the putative RS-like region of NSrp70. After transient transfection, the localization of GFP-tagged NSrp70 mutants was analyzed by confocal microscopy. To better monitor the localization, the fixed cells were also counter stained with endogenous SC35. In contrast to the wild-type (GFP_NSrp70), deletion of amino acids spanning the 439–558 (M1) region appeared to be localized in the cytosol, suggesting that this region included the NLS sequence (Figure 3A). To find the exact NLS sequence, we therefore designed more NSrp70 mutant constructs (Figure 3B). Interestingly, we found that mutant M10 (Δ531–540) localized in the cytosol, suggesting that this region belongs to the NLS sequence of NSrp70. Further point mutations at 536R/A or 537D/A demonstrated that these two amino acids are critical residues for nuclear localization and target the speckle compartment (Figure 3C). To extend these findings with regard to the functional role, we performed in vivo splicing assay with Tra2β1 minigene. M1 (Δ439–558), M8 (Δ531–558) and M10 (Δ531–540), which localize in the cytosol, did not induce alternative splicing (Figure 3D). Interestingly, however, M7 (Δ541–558), which localizes in the nucleus, affected the alternative splice site selection and is comparable to that of the wild-type (Figure 3D). These results demonstrate that nuclear localization by the NLS sequence (531–540), including 536RD537, is important for the functional role of NSrp70. Further, we found that this sequence does not match the known NLS sequence, suggesting that this is a novel NLS sequence.Figure 3.

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