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AG-dependent 3'-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon.

Fu Y, Masuda A, Ito M, Shinmi J, Ohno K - Nucleic Acids Res. (2011)

Bottom Line: RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not.Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing.The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.

ABSTRACT
In pre-mRNA splicing, a conserved AG/G at the 3'-splice site is recognized by U2AF(35). A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E(+1)) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF(35) enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E(+1). Analysis of nine other disease-causing mutations at E(+1) detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing. The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).

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(A) Polypyrimidine stretch and the first nucleotide of an exon in the human genome. The longest stretch of uninterrupted pyrimidines among 25 nt at the 3′-ends of an intron is counted for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (B) Ratios of ‘C’ at position −3 in relation to the first nucleotide of an exon are analyzed for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (C) Preferentially observed nucleotides at the 5′-end of an exon in human. Only wobbling nucleotides are counted in the human genome. (D) Nucleotide frequencies at exonic positions +1 to +8 according to the SELEX data of U2AF35 by Wu and colleagues (12). (E) Effects of ‘TTT’ at exonic positions +3 to +5 in GH1, FECH and EYA1 carrying the patient’s mutation at E+1. Artificially substituted exonic nucleotides are indicated by boxes. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.
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Figure 7: (A) Polypyrimidine stretch and the first nucleotide of an exon in the human genome. The longest stretch of uninterrupted pyrimidines among 25 nt at the 3′-ends of an intron is counted for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (B) Ratios of ‘C’ at position −3 in relation to the first nucleotide of an exon are analyzed for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (C) Preferentially observed nucleotides at the 5′-end of an exon in human. Only wobbling nucleotides are counted in the human genome. (D) Nucleotide frequencies at exonic positions +1 to +8 according to the SELEX data of U2AF35 by Wu and colleagues (12). (E) Effects of ‘TTT’ at exonic positions +3 to +5 in GH1, FECH and EYA1 carrying the patient’s mutation at E+1. Artificially substituted exonic nucleotides are indicated by boxes. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.

Mentions: We next analyzed PPTs of 176 809 introns of the entire human genome. The length of the pyrimidine stretch was shorter when E+1 was the conserved ‘G’ (Figure 7A). This also supports a notion that AG-dependent 3′ ss harboring G at E+1 has a short polypyrimidine stretch (12). In addition, the ratio of ‘C’ at intronic position −3 was lower when E+1 was the conserved ‘G’ (Figure 7B), which suggests that G at E+1 makes C at −3 dispensable for binding to U2AF35, although this is not directly relevant to the length of the PPT.Figure 7.


AG-dependent 3'-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon.

Fu Y, Masuda A, Ito M, Shinmi J, Ohno K - Nucleic Acids Res. (2011)

(A) Polypyrimidine stretch and the first nucleotide of an exon in the human genome. The longest stretch of uninterrupted pyrimidines among 25 nt at the 3′-ends of an intron is counted for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (B) Ratios of ‘C’ at position −3 in relation to the first nucleotide of an exon are analyzed for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (C) Preferentially observed nucleotides at the 5′-end of an exon in human. Only wobbling nucleotides are counted in the human genome. (D) Nucleotide frequencies at exonic positions +1 to +8 according to the SELEX data of U2AF35 by Wu and colleagues (12). (E) Effects of ‘TTT’ at exonic positions +3 to +5 in GH1, FECH and EYA1 carrying the patient’s mutation at E+1. Artificially substituted exonic nucleotides are indicated by boxes. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105431&req=5

Figure 7: (A) Polypyrimidine stretch and the first nucleotide of an exon in the human genome. The longest stretch of uninterrupted pyrimidines among 25 nt at the 3′-ends of an intron is counted for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (B) Ratios of ‘C’ at position −3 in relation to the first nucleotide of an exon are analyzed for 176 809 introns of the human genome. Diamonds represent means and 95% confidence intervals. One-way ANOVA and Fisher’s-multiple range test revealed statistical significance of P < 0.0001. (C) Preferentially observed nucleotides at the 5′-end of an exon in human. Only wobbling nucleotides are counted in the human genome. (D) Nucleotide frequencies at exonic positions +1 to +8 according to the SELEX data of U2AF35 by Wu and colleagues (12). (E) Effects of ‘TTT’ at exonic positions +3 to +5 in GH1, FECH and EYA1 carrying the patient’s mutation at E+1. Artificially substituted exonic nucleotides are indicated by boxes. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.
Mentions: We next analyzed PPTs of 176 809 introns of the entire human genome. The length of the pyrimidine stretch was shorter when E+1 was the conserved ‘G’ (Figure 7A). This also supports a notion that AG-dependent 3′ ss harboring G at E+1 has a short polypyrimidine stretch (12). In addition, the ratio of ‘C’ at intronic position −3 was lower when E+1 was the conserved ‘G’ (Figure 7B), which suggests that G at E+1 makes C at −3 dispensable for binding to U2AF35, although this is not directly relevant to the length of the PPT.Figure 7.

Bottom Line: RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not.Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing.The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).

View Article: PubMed Central - PubMed

Affiliation: Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.

ABSTRACT
In pre-mRNA splicing, a conserved AG/G at the 3'-splice site is recognized by U2AF(35). A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E(+1)) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF(35) enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E(+1). Analysis of nine other disease-causing mutations at E(+1) detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing. The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).

Show MeSH
Related in: MedlinePlus