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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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Exogenous and endogenous NSrp70 are localized within the nucleus as speckles or small punctuate forms. (A, left): COS-7 cells were transfected for 24 h with 2 µg of pEGFP or pEGFP/NSrp70 cDNAs. The cells were stained with DAPI (blue) and phalloidin-TRITC (red), and were observed under a confocal microscope equipped with a 100× objective. (B) Endogenous NSrp70 (green) was detected with a rabbit polyclonal anti-NSrp70 antibody and visualized with an FITC-conjugated anti-rabbit antibody in HEK293T cells. (A and B) The subcellular localization of NSrp70 was confirmed by nuclear fractionation assay of the COS-7 (A, right) and HEK293T (B, right) cell lines. I-κB and PARP (or SC35) were used as cytosolic and nucleoplasmic fraction markers.
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Figure 1: Exogenous and endogenous NSrp70 are localized within the nucleus as speckles or small punctuate forms. (A, left): COS-7 cells were transfected for 24 h with 2 µg of pEGFP or pEGFP/NSrp70 cDNAs. The cells were stained with DAPI (blue) and phalloidin-TRITC (red), and were observed under a confocal microscope equipped with a 100× objective. (B) Endogenous NSrp70 (green) was detected with a rabbit polyclonal anti-NSrp70 antibody and visualized with an FITC-conjugated anti-rabbit antibody in HEK293T cells. (A and B) The subcellular localization of NSrp70 was confirmed by nuclear fractionation assay of the COS-7 (A, right) and HEK293T (B, right) cell lines. I-κB and PARP (or SC35) were used as cytosolic and nucleoplasmic fraction markers.

Mentions: The absence of a connection between NSrp70 and T-cell migration and activation led us to track the localization of NSrp70 in various cell types, as a previous report demonstrated that NSrp70 formed speckle structures in the nucleus (15). We consistently observed typical nuclear speckled patterns when green fluorescent protein (GFP)-tagged NSrp70 cDNA was transfected into COS-7 cells (Figure 1A). High-resolution confocal microscopic analysis revealed either diffused or doughnut-shaped distribution of GFP-tagged NSrp70 in many cells (Supplementary Figure S2A). However, a colocalization study revealed that NSrp70 shows a precise overlap with splicing factor SC35, confirming its localization in nuclear speckles (Supplementary Figure S2B). Endogenous NSrp70 was also detected in the nucleus, but mostly in a diffuse pattern in fixed HEK293T cells (Figure 1B, left). To clarify whether the slightly different pattern of GFP-tagged NSrp70 compared to the endogenous NSrp70 in terms of nuclear localization pattern is due to the artifact of tagged protein, we further used Myc-tagged NSrp70 cDNA in 293T cells, and then co-localization study was performed with SC35. myc-tagged NSrp70 also showed similar pattern as endogenous NSrp70, ranging from the diffuse pattern to the speckle pattern, and was unambiguously overlapped with SC35, therefore suggesting that tagged protein had little effect on NSrp70 localization. Instead, it might be a difference resulted by the direct visualization (GFP) versus indirect visualization after antibody staining. Localization of NSrp70 was further confirmed by nuclear fractionation assay with COS-7 and HEK293T cells (Figure 1A and B, right). Sequence alignment of NSrp70 orthologs demonstrated a highly conserved homology with other species (Supplementary Figure S3).Figure 1.


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)

Exogenous and endogenous NSrp70 are localized within the nucleus as speckles or small punctuate forms. (A, left): COS-7 cells were transfected for 24 h with 2 µg of pEGFP or pEGFP/NSrp70 cDNAs. The cells were stained with DAPI (blue) and phalloidin-TRITC (red), and were observed under a confocal microscope equipped with a 100× objective. (B) Endogenous NSrp70 (green) was detected with a rabbit polyclonal anti-NSrp70 antibody and visualized with an FITC-conjugated anti-rabbit antibody in HEK293T cells. (A and B) The subcellular localization of NSrp70 was confirmed by nuclear fractionation assay of the COS-7 (A, right) and HEK293T (B, right) cell lines. I-κB and PARP (or SC35) were used as cytosolic and nucleoplasmic fraction markers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105421&req=5

Figure 1: Exogenous and endogenous NSrp70 are localized within the nucleus as speckles or small punctuate forms. (A, left): COS-7 cells were transfected for 24 h with 2 µg of pEGFP or pEGFP/NSrp70 cDNAs. The cells were stained with DAPI (blue) and phalloidin-TRITC (red), and were observed under a confocal microscope equipped with a 100× objective. (B) Endogenous NSrp70 (green) was detected with a rabbit polyclonal anti-NSrp70 antibody and visualized with an FITC-conjugated anti-rabbit antibody in HEK293T cells. (A and B) The subcellular localization of NSrp70 was confirmed by nuclear fractionation assay of the COS-7 (A, right) and HEK293T (B, right) cell lines. I-κB and PARP (or SC35) were used as cytosolic and nucleoplasmic fraction markers.
Mentions: The absence of a connection between NSrp70 and T-cell migration and activation led us to track the localization of NSrp70 in various cell types, as a previous report demonstrated that NSrp70 formed speckle structures in the nucleus (15). We consistently observed typical nuclear speckled patterns when green fluorescent protein (GFP)-tagged NSrp70 cDNA was transfected into COS-7 cells (Figure 1A). High-resolution confocal microscopic analysis revealed either diffused or doughnut-shaped distribution of GFP-tagged NSrp70 in many cells (Supplementary Figure S2A). However, a colocalization study revealed that NSrp70 shows a precise overlap with splicing factor SC35, confirming its localization in nuclear speckles (Supplementary Figure S2B). Endogenous NSrp70 was also detected in the nucleus, but mostly in a diffuse pattern in fixed HEK293T cells (Figure 1B, left). To clarify whether the slightly different pattern of GFP-tagged NSrp70 compared to the endogenous NSrp70 in terms of nuclear localization pattern is due to the artifact of tagged protein, we further used Myc-tagged NSrp70 cDNA in 293T cells, and then co-localization study was performed with SC35. myc-tagged NSrp70 also showed similar pattern as endogenous NSrp70, ranging from the diffuse pattern to the speckle pattern, and was unambiguously overlapped with SC35, therefore suggesting that tagged protein had little effect on NSrp70 localization. Instead, it might be a difference resulted by the direct visualization (GFP) versus indirect visualization after antibody staining. Localization of NSrp70 was further confirmed by nuclear fractionation assay with COS-7 and HEK293T cells (Figure 1A and B, right). Sequence alignment of NSrp70 orthologs demonstrated a highly conserved homology with other species (Supplementary Figure S3).Figure 1.

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