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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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Purification of the pre-spliceosomal E complex formed on hF1γ exon 9–10 pre-mRNA in vitro. (A) The schematic representation of pre-mRNAs, CDC14-15, ftz, hF1γ3GCAUG and hF1γΔGCAUG, fused with two MS2-binding sites at the 3′ exons. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). Average and standard deviation (SD) of the ratio of U1 snRNA to U2 snRNA from three independent experiments are shown at the bottom. (C) Western blotting of the purified E complexes on the pre-mRNAs using U1-70K and U2AF65 antibodies (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (D) The sequences of 5′ splice sites of four kinds of pre-mRNAs as shown in (A). Capital and lowercase letters correspond to exonic and intronic residues, respectively. The nucleotide different from the consensus site is shown in bold.
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Figure 1: Purification of the pre-spliceosomal E complex formed on hF1γ exon 9–10 pre-mRNA in vitro. (A) The schematic representation of pre-mRNAs, CDC14-15, ftz, hF1γ3GCAUG and hF1γΔGCAUG, fused with two MS2-binding sites at the 3′ exons. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). Average and standard deviation (SD) of the ratio of U1 snRNA to U2 snRNA from three independent experiments are shown at the bottom. (C) Western blotting of the purified E complexes on the pre-mRNAs using U1-70K and U2AF65 antibodies (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (D) The sequences of 5′ splice sites of four kinds of pre-mRNAs as shown in (A). Capital and lowercase letters correspond to exonic and intronic residues, respectively. The nucleotide different from the consensus site is shown in bold.

Mentions: In our previous study, we showed that Fox-1 induces the hF1γ exon 9 skipping by interfering with the pre-spliceosomal E complex formation on intron 9 (exon 9–10) (13). To further clarify the mechanism, we performed affinity purification of the E complex formed on hF1γ exon 9–10 pre-mRNA fused with two MS2 binding sites at the terminus of 3′ exon (Figure 1A). The hF1γ3GCAUG pre-mRNA contains a portion of the preceding intron 8 with three copies of the GCAUG element (Fox-1 binding element). Note that the branch point in intron 8 was disrupted by base-substitution mutations. The GCAUG elements were deleted in hF1γΔGCAUG pre-mRNA (Figure 1A). We also used CDC14-15 (chicken δ crystallin exon 14–15) pre-mRNA with MS2-binding sites as a control substrate, which is often used for in vitro splicing analyses (20,21). Under our in vitro splicing conditions, hF1γ pre-mRNAs were spliced as efficiently as CDC14-15 (see Supplementary Figure S1).Figure 1.


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)

Purification of the pre-spliceosomal E complex formed on hF1γ exon 9–10 pre-mRNA in vitro. (A) The schematic representation of pre-mRNAs, CDC14-15, ftz, hF1γ3GCAUG and hF1γΔGCAUG, fused with two MS2-binding sites at the 3′ exons. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). Average and standard deviation (SD) of the ratio of U1 snRNA to U2 snRNA from three independent experiments are shown at the bottom. (C) Western blotting of the purified E complexes on the pre-mRNAs using U1-70K and U2AF65 antibodies (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (D) The sequences of 5′ splice sites of four kinds of pre-mRNAs as shown in (A). Capital and lowercase letters correspond to exonic and intronic residues, respectively. The nucleotide different from the consensus site is shown in bold.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Purification of the pre-spliceosomal E complex formed on hF1γ exon 9–10 pre-mRNA in vitro. (A) The schematic representation of pre-mRNAs, CDC14-15, ftz, hF1γ3GCAUG and hF1γΔGCAUG, fused with two MS2-binding sites at the 3′ exons. Boxes and lines represent exons and introns, respectively. The Fox-1 binding element GCAUG is shown as a closed circle. (B) Northern blotting of the purified E complexes with U1 and U2 snRNA probes (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). Average and standard deviation (SD) of the ratio of U1 snRNA to U2 snRNA from three independent experiments are shown at the bottom. (C) Western blotting of the purified E complexes on the pre-mRNAs using U1-70K and U2AF65 antibodies (lanes 5–8) and aliquots of the reaction mixtures (lanes 1–4). (D) The sequences of 5′ splice sites of four kinds of pre-mRNAs as shown in (A). Capital and lowercase letters correspond to exonic and intronic residues, respectively. The nucleotide different from the consensus site is shown in bold.
Mentions: In our previous study, we showed that Fox-1 induces the hF1γ exon 9 skipping by interfering with the pre-spliceosomal E complex formation on intron 9 (exon 9–10) (13). To further clarify the mechanism, we performed affinity purification of the E complex formed on hF1γ exon 9–10 pre-mRNA fused with two MS2 binding sites at the terminus of 3′ exon (Figure 1A). The hF1γ3GCAUG pre-mRNA contains a portion of the preceding intron 8 with three copies of the GCAUG element (Fox-1 binding element). Note that the branch point in intron 8 was disrupted by base-substitution mutations. The GCAUG elements were deleted in hF1γΔGCAUG pre-mRNA (Figure 1A). We also used CDC14-15 (chicken δ crystallin exon 14–15) pre-mRNA with MS2-binding sites as a control substrate, which is often used for in vitro splicing analyses (20,21). Under our in vitro splicing conditions, hF1γ pre-mRNAs were spliced as efficiently as CDC14-15 (see Supplementary Figure S1).Figure 1.

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