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U1-independent pre-mRNA splicing contributes to the regulation of alternative splicing.

Fukumura K, Taniguchi I, Sakamoto H, Ohno M, Inoue K - Nucleic Acids Res. (2009)

Bottom Line: Moreover, hF1gamma intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts.Finally, hF1gamma exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one.Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Science, Kobe University, Nadaku, Kobe, Japan.

ABSTRACT
U1 snRNP plays a crucial role in the 5' splice site recognition during splicing. Here we report the first example of naturally occurring U1-independent U2-type splicing in humans. The U1 components were not included in the pre-spliceosomal E complex formed on the human F1gamma (hF1gamma) intron 9 in vitro. Moreover, hF1gamma intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts. Finally, hF1gamma exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one. Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

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U1-independent splicing is indispensable for the regulation of alternative splicing of hF1γ by Fox-1. (A) The schematic representation of wild-type (WT) and mutant (mtU1) hF1γ mini-genes (left) and the suppressor U1 snRNP (right). The mutations introduced into the 5′ splice site of exon 9 are underlined. Closed circles indicate the Fox-1 binding element. (B) Transfection assay of hF1γ WT and 5′SSmt mini-genes co-expressed with pCS2+MT vector or pCS+MT-Fox-1 in HeLa cells. The left panel shows spliced products amplified by the oligonucleotides annealed with exons 8 and 10. The middle and right panels show the splicing reaction between exons 8 and 9 and between exons 9 and 10. Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed four times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (C) Suppressor U1 snRNA experiments in HeLa cells. The wild-type or mutant U1 snRNA was co-expressed with wild-type hF1γ mini-gene and pCS2+MT vector or pCS+MT-Fox-1. The spliced products were analyzed by RT-PCR using the oligonucleotides annealed with exons 8 and 10 (left panel), exons 8 and 9 (middle panel) and exons 9 and 10 (right panel). Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed three times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (D) Western blotting of cell extracts using the anti-Myc antibody (upper panel) and anti-U2AF65 antibody as a loading control (lower panel). Molecular size markers are shown on the right.
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Figure 5: U1-independent splicing is indispensable for the regulation of alternative splicing of hF1γ by Fox-1. (A) The schematic representation of wild-type (WT) and mutant (mtU1) hF1γ mini-genes (left) and the suppressor U1 snRNP (right). The mutations introduced into the 5′ splice site of exon 9 are underlined. Closed circles indicate the Fox-1 binding element. (B) Transfection assay of hF1γ WT and 5′SSmt mini-genes co-expressed with pCS2+MT vector or pCS+MT-Fox-1 in HeLa cells. The left panel shows spliced products amplified by the oligonucleotides annealed with exons 8 and 10. The middle and right panels show the splicing reaction between exons 8 and 9 and between exons 9 and 10. Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed four times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (C) Suppressor U1 snRNA experiments in HeLa cells. The wild-type or mutant U1 snRNA was co-expressed with wild-type hF1γ mini-gene and pCS2+MT vector or pCS+MT-Fox-1. The spliced products were analyzed by RT-PCR using the oligonucleotides annealed with exons 8 and 10 (left panel), exons 8 and 9 (middle panel) and exons 9 and 10 (right panel). Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed three times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (D) Western blotting of cell extracts using the anti-Myc antibody (upper panel) and anti-U2AF65 antibody as a loading control (lower panel). Molecular size markers are shown on the right.

Mentions: First, we performed transfection experiments using a mutant hF1γ mini-gene, hF1γS3GCAUG (5′SSmt), in which the 5′ splice site of intron 9 had been substituted to that of CDC14-15, namely the U1-dependent splice site (Figure 5A). Transfection assay of wild-type hF1γ mini-gene into HeLa cells showed that Fox-1 proteins promoted exon 9 exclusion as described in our previous report (13) (Figure 5B, lanes 1 and 2). It has been shown that endogenous Fox-1 is undetectable in HeLa cells (11). In contrast, when the mutant mini-gene hF1γS3GCAUG (5′SSmt) containing the U1-dependent splice site was transfected, exon 9 exclusion was barely observed even in the presence of Fox-1 (Figure 5B, lanes 3 and 4). Note that the splicing efficiencies of introns 8 and 9 were not largely affected by the mutation into the splice site in the absence of Fox-1 (lanes 5, 7, 9 and 11). Importantly, when the Fox-1 protein was co-expressed (Figure 5D), splicing of the downstream intron 9 (E9–E10) of wild-type hF1γ mini-gene was inhibited by Fox-1 as expected (13), whereas that of the mutant mini-gene hF1γS3GCAUG (5′SSmt) was not (Figure 5B, lanes 9–12). Irrespective of the mutation into the 5′ splice site, splicing of intron 8 (E8–E9) was not affected by the Fox-1 expression (lanes 5–8). In addition, we confirmed that the expressed Fox-1 protein was properly localized to the nucleus (data not shown).Figure 5.


U1-independent pre-mRNA splicing contributes to the regulation of alternative splicing.

Fukumura K, Taniguchi I, Sakamoto H, Ohno M, Inoue K - Nucleic Acids Res. (2009)

U1-independent splicing is indispensable for the regulation of alternative splicing of hF1γ by Fox-1. (A) The schematic representation of wild-type (WT) and mutant (mtU1) hF1γ mini-genes (left) and the suppressor U1 snRNP (right). The mutations introduced into the 5′ splice site of exon 9 are underlined. Closed circles indicate the Fox-1 binding element. (B) Transfection assay of hF1γ WT and 5′SSmt mini-genes co-expressed with pCS2+MT vector or pCS+MT-Fox-1 in HeLa cells. The left panel shows spliced products amplified by the oligonucleotides annealed with exons 8 and 10. The middle and right panels show the splicing reaction between exons 8 and 9 and between exons 9 and 10. Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed four times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (C) Suppressor U1 snRNA experiments in HeLa cells. The wild-type or mutant U1 snRNA was co-expressed with wild-type hF1γ mini-gene and pCS2+MT vector or pCS+MT-Fox-1. The spliced products were analyzed by RT-PCR using the oligonucleotides annealed with exons 8 and 10 (left panel), exons 8 and 9 (middle panel) and exons 9 and 10 (right panel). Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed three times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (D) Western blotting of cell extracts using the anti-Myc antibody (upper panel) and anti-U2AF65 antibody as a loading control (lower panel). Molecular size markers are shown on the right.
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Figure 5: U1-independent splicing is indispensable for the regulation of alternative splicing of hF1γ by Fox-1. (A) The schematic representation of wild-type (WT) and mutant (mtU1) hF1γ mini-genes (left) and the suppressor U1 snRNP (right). The mutations introduced into the 5′ splice site of exon 9 are underlined. Closed circles indicate the Fox-1 binding element. (B) Transfection assay of hF1γ WT and 5′SSmt mini-genes co-expressed with pCS2+MT vector or pCS+MT-Fox-1 in HeLa cells. The left panel shows spliced products amplified by the oligonucleotides annealed with exons 8 and 10. The middle and right panels show the splicing reaction between exons 8 and 9 and between exons 9 and 10. Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed four times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (C) Suppressor U1 snRNA experiments in HeLa cells. The wild-type or mutant U1 snRNA was co-expressed with wild-type hF1γ mini-gene and pCS2+MT vector or pCS+MT-Fox-1. The spliced products were analyzed by RT-PCR using the oligonucleotides annealed with exons 8 and 10 (left panel), exons 8 and 9 (middle panel) and exons 9 and 10 (right panel). Positions of splicing products and unspliced transcripts are schematically shown on the right. All experiments were independently performed three times. Average and standard deviation of exon 9 exclusion and splicing efficiency are shown at the bottom of each lane. (D) Western blotting of cell extracts using the anti-Myc antibody (upper panel) and anti-U2AF65 antibody as a loading control (lower panel). Molecular size markers are shown on the right.
Mentions: First, we performed transfection experiments using a mutant hF1γ mini-gene, hF1γS3GCAUG (5′SSmt), in which the 5′ splice site of intron 9 had been substituted to that of CDC14-15, namely the U1-dependent splice site (Figure 5A). Transfection assay of wild-type hF1γ mini-gene into HeLa cells showed that Fox-1 proteins promoted exon 9 exclusion as described in our previous report (13) (Figure 5B, lanes 1 and 2). It has been shown that endogenous Fox-1 is undetectable in HeLa cells (11). In contrast, when the mutant mini-gene hF1γS3GCAUG (5′SSmt) containing the U1-dependent splice site was transfected, exon 9 exclusion was barely observed even in the presence of Fox-1 (Figure 5B, lanes 3 and 4). Note that the splicing efficiencies of introns 8 and 9 were not largely affected by the mutation into the splice site in the absence of Fox-1 (lanes 5, 7, 9 and 11). Importantly, when the Fox-1 protein was co-expressed (Figure 5D), splicing of the downstream intron 9 (E9–E10) of wild-type hF1γ mini-gene was inhibited by Fox-1 as expected (13), whereas that of the mutant mini-gene hF1γS3GCAUG (5′SSmt) was not (Figure 5B, lanes 9–12). Irrespective of the mutation into the 5′ splice site, splicing of intron 8 (E8–E9) was not affected by the Fox-1 expression (lanes 5–8). In addition, we confirmed that the expressed Fox-1 protein was properly localized to the nucleus (data not shown).Figure 5.

Bottom Line: Moreover, hF1gamma intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts.Finally, hF1gamma exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one.Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Science, Kobe University, Nadaku, Kobe, Japan.

ABSTRACT
U1 snRNP plays a crucial role in the 5' splice site recognition during splicing. Here we report the first example of naturally occurring U1-independent U2-type splicing in humans. The U1 components were not included in the pre-spliceosomal E complex formed on the human F1gamma (hF1gamma) intron 9 in vitro. Moreover, hF1gamma intron 9 was efficiently spliced even in U1-disrupted Xenopus oocytes as well as in U1-inactivated HeLa nuclear extracts. Finally, hF1gamma exon 9 skipping induced by an alternative splicing regulator Fox-1 was impaired when intron 9 was changed to the U1-dependent one. Our results suggest that U1-independent splicing contributes to the regulation of alternative splicing of a class of pre-mRNAs.

Show MeSH