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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 influences alternative splice site selection of several minigenes in different cell types. (A) Promotion of exon v2 exclusion on Tra2β1 and exon v5 inclusion on CD44 minigene by NSrp70. HEK293T cells were cotransfected with increasing amounts of pEGFP/NSrp70 (0–2 µg) and 2 µg of the minigene. Parental vector (0–2 µg) was added to ensure that similar amounts of cDNAs were transfected. Exon inclusion or exclusion was determined by RT–PCR (top), as described in ‘Materials and Methods’ section. The ratio of exclusion or inclusion of Tra2β1 or CD44 minigene was shown as a histogram (middle). Dose-dependent expression of GFP_NSrp70 and GFP was confirmed by western blotting (bottom). (B) NSrp70 promotes exon v6 inclusion on Fas minigene in HEK293T or Jurkat T cells. Two micrograms of Fas minigene was transfected with 2 µg of pEGFP/NSrp70 or pEGFP into HEK293T or Jurkat T cells. Exon inclusion or exclusion was determined by RT–PCR. Note: P, parent; EV, pEGFP vector; NSrp70, pEGFP/NSrp70. Asterisks denote an uncharacterized PCR product (A and B). (C) Interference of NSrp70 by shRNA decreases exon v6 inclusion in Jurkat T cells. psiLv-H1/scramble or psiLV-H1/shNSrp70 (CCDC55) vector was infected to Jurkat T cells by the lentiviral system. Exon inclusion or exclusion was determined by RT–PCR. Note: M, 100 bp DNA marker; SR, psiLv-H1/scramble, NSrp70, psiLV-H1/shNSrp70 infection. The ratio of exon inclusion or exclusion splicing forms was analyzed by ImageJ. Each experiment was repeated at least three times to confirm the reproducibility of data. (D and E) mRNA of minigenes interacts to NSrp70. HEK293T cells were transfected with (D) or without (E) plasmid expressing myc_NSrp70 and indicated minigene and then immunoprecipitated with anti-Myc (D) or anti-NSrp70 antibody (E). The RNAs in the immunoprecipitates were then analyzed by RT–PCR using primers specific for the CD44, Fas or Tra2β1 transcripts of minigenes. Ten microliter of the supernatant of total lysate was used for total input sample.
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Figure 2: NSrp70 influences alternative splice site selection of several minigenes in different cell types. (A) Promotion of exon v2 exclusion on Tra2β1 and exon v5 inclusion on CD44 minigene by NSrp70. HEK293T cells were cotransfected with increasing amounts of pEGFP/NSrp70 (0–2 µg) and 2 µg of the minigene. Parental vector (0–2 µg) was added to ensure that similar amounts of cDNAs were transfected. Exon inclusion or exclusion was determined by RT–PCR (top), as described in ‘Materials and Methods’ section. The ratio of exclusion or inclusion of Tra2β1 or CD44 minigene was shown as a histogram (middle). Dose-dependent expression of GFP_NSrp70 and GFP was confirmed by western blotting (bottom). (B) NSrp70 promotes exon v6 inclusion on Fas minigene in HEK293T or Jurkat T cells. Two micrograms of Fas minigene was transfected with 2 µg of pEGFP/NSrp70 or pEGFP into HEK293T or Jurkat T cells. Exon inclusion or exclusion was determined by RT–PCR. Note: P, parent; EV, pEGFP vector; NSrp70, pEGFP/NSrp70. Asterisks denote an uncharacterized PCR product (A and B). (C) Interference of NSrp70 by shRNA decreases exon v6 inclusion in Jurkat T cells. psiLv-H1/scramble or psiLV-H1/shNSrp70 (CCDC55) vector was infected to Jurkat T cells by the lentiviral system. Exon inclusion or exclusion was determined by RT–PCR. Note: M, 100 bp DNA marker; SR, psiLv-H1/scramble, NSrp70, psiLV-H1/shNSrp70 infection. The ratio of exon inclusion or exclusion splicing forms was analyzed by ImageJ. Each experiment was repeated at least three times to confirm the reproducibility of data. (D and E) mRNA of minigenes interacts to NSrp70. HEK293T cells were transfected with (D) or without (E) plasmid expressing myc_NSrp70 and indicated minigene and then immunoprecipitated with anti-Myc (D) or anti-NSrp70 antibody (E). The RNAs in the immunoprecipitates were then analyzed by RT–PCR using primers specific for the CD44, Fas or Tra2β1 transcripts of minigenes. Ten microliter of the supernatant of total lysate was used for total input sample.

Mentions: Based on the results obtained from sequence alignment with other SR or SR-related proteins, we next conducted in vivo splicing assays using minigenes to test whether NSrp70 behaves like a typical SR protein i.e. whether it can activate pre-mRNA splicing or regulate alternative splicing in a concentration-dependent manner (5,19). To this end, Tra2β1 or CD44 minigene was transfected into HEK293T cells with increasing amounts of pEGFP/NSrp70. RT–PCR was performed with primers directed at sequences located in the flanking constitutive exons. As shown in Figure 2A, we found that addition of NSrp70 in small increment from 0.5 to 2 µg resulted in an increased exon v2 exclusion from 69 to 92% in Tra2β1 minigene and exon v5 inclusion from 39 to 65% in CD44 minigene. As a control, cotransfection of Tra2β1 or CD44 with an empty vector, pEGFP-C1, did not change the splicing pattern, demonstrating that the splicing reaction and involvement of NSrp70 in splicing modulation were not an artifact. Western blot data represent the protein amounts after transfection of GFP and GFP_NSrp70 in HEK293T cells (Figure 2A).Figure 2.


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 influences alternative splice site selection of several minigenes in different cell types. (A) Promotion of exon v2 exclusion on Tra2β1 and exon v5 inclusion on CD44 minigene by NSrp70. HEK293T cells were cotransfected with increasing amounts of pEGFP/NSrp70 (0–2 µg) and 2 µg of the minigene. Parental vector (0–2 µg) was added to ensure that similar amounts of cDNAs were transfected. Exon inclusion or exclusion was determined by RT–PCR (top), as described in ‘Materials and Methods’ section. The ratio of exclusion or inclusion of Tra2β1 or CD44 minigene was shown as a histogram (middle). Dose-dependent expression of GFP_NSrp70 and GFP was confirmed by western blotting (bottom). (B) NSrp70 promotes exon v6 inclusion on Fas minigene in HEK293T or Jurkat T cells. Two micrograms of Fas minigene was transfected with 2 µg of pEGFP/NSrp70 or pEGFP into HEK293T or Jurkat T cells. Exon inclusion or exclusion was determined by RT–PCR. Note: P, parent; EV, pEGFP vector; NSrp70, pEGFP/NSrp70. Asterisks denote an uncharacterized PCR product (A and B). (C) Interference of NSrp70 by shRNA decreases exon v6 inclusion in Jurkat T cells. psiLv-H1/scramble or psiLV-H1/shNSrp70 (CCDC55) vector was infected to Jurkat T cells by the lentiviral system. Exon inclusion or exclusion was determined by RT–PCR. Note: M, 100 bp DNA marker; SR, psiLv-H1/scramble, NSrp70, psiLV-H1/shNSrp70 infection. The ratio of exon inclusion or exclusion splicing forms was analyzed by ImageJ. Each experiment was repeated at least three times to confirm the reproducibility of data. (D and E) mRNA of minigenes interacts to NSrp70. HEK293T cells were transfected with (D) or without (E) plasmid expressing myc_NSrp70 and indicated minigene and then immunoprecipitated with anti-Myc (D) or anti-NSrp70 antibody (E). The RNAs in the immunoprecipitates were then analyzed by RT–PCR using primers specific for the CD44, Fas or Tra2β1 transcripts of minigenes. Ten microliter of the supernatant of total lysate was used for total input sample.
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Figure 2: NSrp70 influences alternative splice site selection of several minigenes in different cell types. (A) Promotion of exon v2 exclusion on Tra2β1 and exon v5 inclusion on CD44 minigene by NSrp70. HEK293T cells were cotransfected with increasing amounts of pEGFP/NSrp70 (0–2 µg) and 2 µg of the minigene. Parental vector (0–2 µg) was added to ensure that similar amounts of cDNAs were transfected. Exon inclusion or exclusion was determined by RT–PCR (top), as described in ‘Materials and Methods’ section. The ratio of exclusion or inclusion of Tra2β1 or CD44 minigene was shown as a histogram (middle). Dose-dependent expression of GFP_NSrp70 and GFP was confirmed by western blotting (bottom). (B) NSrp70 promotes exon v6 inclusion on Fas minigene in HEK293T or Jurkat T cells. Two micrograms of Fas minigene was transfected with 2 µg of pEGFP/NSrp70 or pEGFP into HEK293T or Jurkat T cells. Exon inclusion or exclusion was determined by RT–PCR. Note: P, parent; EV, pEGFP vector; NSrp70, pEGFP/NSrp70. Asterisks denote an uncharacterized PCR product (A and B). (C) Interference of NSrp70 by shRNA decreases exon v6 inclusion in Jurkat T cells. psiLv-H1/scramble or psiLV-H1/shNSrp70 (CCDC55) vector was infected to Jurkat T cells by the lentiviral system. Exon inclusion or exclusion was determined by RT–PCR. Note: M, 100 bp DNA marker; SR, psiLv-H1/scramble, NSrp70, psiLV-H1/shNSrp70 infection. The ratio of exon inclusion or exclusion splicing forms was analyzed by ImageJ. Each experiment was repeated at least three times to confirm the reproducibility of data. (D and E) mRNA of minigenes interacts to NSrp70. HEK293T cells were transfected with (D) or without (E) plasmid expressing myc_NSrp70 and indicated minigene and then immunoprecipitated with anti-Myc (D) or anti-NSrp70 antibody (E). The RNAs in the immunoprecipitates were then analyzed by RT–PCR using primers specific for the CD44, Fas or Tra2β1 transcripts of minigenes. Ten microliter of the supernatant of total lysate was used for total input sample.
Mentions: Based on the results obtained from sequence alignment with other SR or SR-related proteins, we next conducted in vivo splicing assays using minigenes to test whether NSrp70 behaves like a typical SR protein i.e. whether it can activate pre-mRNA splicing or regulate alternative splicing in a concentration-dependent manner (5,19). To this end, Tra2β1 or CD44 minigene was transfected into HEK293T cells with increasing amounts of pEGFP/NSrp70. RT–PCR was performed with primers directed at sequences located in the flanking constitutive exons. As shown in Figure 2A, we found that addition of NSrp70 in small increment from 0.5 to 2 µg resulted in an increased exon v2 exclusion from 69 to 92% in Tra2β1 minigene and exon v5 inclusion from 39 to 65% in CD44 minigene. As a control, cotransfection of Tra2β1 or CD44 with an empty vector, pEGFP-C1, did not change the splicing pattern, demonstrating that the splicing reaction and involvement of NSrp70 in splicing modulation were not an artifact. Western blot data represent the protein amounts after transfection of GFP and GFP_NSrp70 in HEK293T cells (Figure 2A).Figure 2.

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