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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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Role of the 5′ splice site sequence in U1-independent splicing. (A) Base-substitutions introduced into the 5′ splice site of hF1γ3GCAUG pre-mRNA are shown (–3G > C and +5a > g). Capital and lowercase letters correspond to exonic and intronic nucleotides, respectively. 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). (C) Splicing reaction of hF1γ3GCAUG (lanes 1–3) and hF1γmt5′SS (lanes 4–6). These pre-mRNAs were incubated under the standard in vitro splicing condition for indicated time above each lane. Pre-mRNA, intermediates and splicing products are indicated schematically on the right.
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Figure 4: Role of the 5′ splice site sequence in U1-independent splicing. (A) Base-substitutions introduced into the 5′ splice site of hF1γ3GCAUG pre-mRNA are shown (–3G > C and +5a > g). Capital and lowercase letters correspond to exonic and intronic nucleotides, respectively. 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). (C) Splicing reaction of hF1γ3GCAUG (lanes 1–3) and hF1γmt5′SS (lanes 4–6). These pre-mRNAs were incubated under the standard in vitro splicing condition for indicated time above each lane. Pre-mRNA, intermediates and splicing products are indicated schematically on the right.

Mentions: The hF1γ intron 9 possesses the typical GT-AG boundaries, although the 5′ splice site has a suboptimal sequence as compared with the consensus sequence, CAG/guaagu (Figure 1D). The 5′ splice site sequence of intron 9 has a conserved sequence at least in humans, mice and bovine (25). To examine if the 5′ splice site sequence is crucial for U1-independent splicing, two base-substitutions were introduced (–3G>C and +5a>g) to change the hF1γ 5′ splice site sequence to that of CDC14-15 (Figure 4A). The mutant was spliced efficiently as the parent substrate in vitro (Figure 4C). Northern blotting showed that the base-substitutions into the 5′ splice site drastically led to the normal E complex formation containing U1 snRNP (Figure 4B). These results strongly suggested that the positions at –3C and +5G of 5′ splice site are important for U1 snRNP binding.Figure 4.


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)

Role of the 5′ splice site sequence in U1-independent splicing. (A) Base-substitutions introduced into the 5′ splice site of hF1γ3GCAUG pre-mRNA are shown (–3G > C and +5a > g). Capital and lowercase letters correspond to exonic and intronic nucleotides, respectively. 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). (C) Splicing reaction of hF1γ3GCAUG (lanes 1–3) and hF1γmt5′SS (lanes 4–6). These pre-mRNAs were incubated under the standard in vitro splicing condition for indicated time above each lane. Pre-mRNA, intermediates and splicing products are indicated schematically on the right.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Role of the 5′ splice site sequence in U1-independent splicing. (A) Base-substitutions introduced into the 5′ splice site of hF1γ3GCAUG pre-mRNA are shown (–3G > C and +5a > g). Capital and lowercase letters correspond to exonic and intronic nucleotides, respectively. 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). (C) Splicing reaction of hF1γ3GCAUG (lanes 1–3) and hF1γmt5′SS (lanes 4–6). These pre-mRNAs were incubated under the standard in vitro splicing condition for indicated time above each lane. Pre-mRNA, intermediates and splicing products are indicated schematically on the right.
Mentions: The hF1γ intron 9 possesses the typical GT-AG boundaries, although the 5′ splice site has a suboptimal sequence as compared with the consensus sequence, CAG/guaagu (Figure 1D). The 5′ splice site sequence of intron 9 has a conserved sequence at least in humans, mice and bovine (25). To examine if the 5′ splice site sequence is crucial for U1-independent splicing, two base-substitutions were introduced (–3G>C and +5a>g) to change the hF1γ 5′ splice site sequence to that of CDC14-15 (Figure 4A). The mutant was spliced efficiently as the parent substrate in vitro (Figure 4C). Northern blotting showed that the base-substitutions into the 5′ splice site drastically led to the normal E complex formation containing U1 snRNP (Figure 4B). These results strongly suggested that the positions at –3C and +5G of 5′ splice site are important for U1 snRNP binding.Figure 4.

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