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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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Deletion of the coiled-coil domain results in loss of alternative splicing activity. (A) Schematic diagram of deletion mutants of NSrp70 (left) and their subcellular localization with SC35 in HEK293T cells (right). (B) 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 4: Deletion of the coiled-coil domain results in loss of alternative splicing activity. (A) Schematic diagram of deletion mutants of NSrp70 (left) and their subcellular localization with SC35 in HEK293T cells (right). (B) 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: SR proteins contain one or two RRMs containing the signature sequences RDAEDA, RDADDA and SWQDLKD (23,24). Splice-site selection is determined by the nature of the RRM (25). As NSrp70 also contains the highly conserved RDAEDA sequence between amino acids 47 and 70 at the N terminus (Supplementary Figure S3), we investigated the role of this sequence in terms of alternative splicing activity. As the N terminus also contains a coiled-coil domain in amino acids 106–161, we also tested whether this region corresponds to the splicing activity of NSrp70. To this end, we designed a series of N-terminal deletion constructs and investigated their splicing activities for Tra2β1 minigene in HEK293T cells (Figure 4A). Interestingly, although all of the deletion mutants (M11–M13) showed nuclear speckle localization, M12 (Δ1–170), a deletion mutant of the coiled-coil domain, but not M11 (Δ1–105), completely lost alternative splice site selection of pre-mRNA of Tra2β1 (Figure 4B), suggesting that the coiled-coil domain (106–170 amino acids) is critical for alternative splicing activity of NSrp70. All expressed proteins accumulated to similar levels in transfected HEK293T cells, as verified by western blot analysis of whole cell lysates (Figure 4B, right).Figure 4.


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)

Deletion of the coiled-coil domain results in loss of alternative splicing activity. (A) Schematic diagram of deletion mutants of NSrp70 (left) and their subcellular localization with SC35 in HEK293T cells (right). (B) 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Deletion of the coiled-coil domain results in loss of alternative splicing activity. (A) Schematic diagram of deletion mutants of NSrp70 (left) and their subcellular localization with SC35 in HEK293T cells (right). (B) 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: SR proteins contain one or two RRMs containing the signature sequences RDAEDA, RDADDA and SWQDLKD (23,24). Splice-site selection is determined by the nature of the RRM (25). As NSrp70 also contains the highly conserved RDAEDA sequence between amino acids 47 and 70 at the N terminus (Supplementary Figure S3), we investigated the role of this sequence in terms of alternative splicing activity. As the N terminus also contains a coiled-coil domain in amino acids 106–161, we also tested whether this region corresponds to the splicing activity of NSrp70. To this end, we designed a series of N-terminal deletion constructs and investigated their splicing activities for Tra2β1 minigene in HEK293T cells (Figure 4A). Interestingly, although all of the deletion mutants (M11–M13) showed nuclear speckle localization, M12 (Δ1–170), a deletion mutant of the coiled-coil domain, but not M11 (Δ1–105), completely lost alternative splice site selection of pre-mRNA of Tra2β1 (Figure 4B), suggesting that the coiled-coil domain (106–170 amino acids) is critical for alternative splicing activity of NSrp70. All expressed proteins accumulated to similar levels in transfected HEK293T cells, as verified by western blot analysis of whole cell lysates (Figure 4B, right).Figure 4.

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