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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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Related in: MedlinePlus

Recapitulation of normal and aberrant splicing of five genes. (A) Nucleotide sequences at the intron/exon junctions of five analyzed genes. Putative BPS is underlined. PPT is shown by a bracket. Mutant nucleotides are indicated at E+1. (B) RT–PCR of minigenes expressed in HEK293 cells carrying the wild-type (WT) or patient’s (PT) nucleotide. The mutations cause exon skipping in GH1, FECH and EYA1, but not in LPL and HEXA. 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 1: Recapitulation of normal and aberrant splicing of five genes. (A) Nucleotide sequences at the intron/exon junctions of five analyzed genes. Putative BPS is underlined. PPT is shown by a bracket. Mutant nucleotides are indicated at E+1. (B) RT–PCR of minigenes expressed in HEK293 cells carrying the wild-type (WT) or patient’s (PT) nucleotide. The mutations cause exon skipping in GH1, FECH and EYA1, but not in LPL and HEXA. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.

Mentions: We first constructed minigenes of GH1, FECH, EYA1, LPL and HEXA, and introduced a previously reported disease-causing mutation at E+1 (Figure 1A). These minigenes successfully recapitulated normal and aberrant splicings: mutations in GH1, FECH and EYA1 caused exon skipping, whereas those in LPL or HEXA did not (Figure 1B).Figure 1.


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)

Recapitulation of normal and aberrant splicing of five genes. (A) Nucleotide sequences at the intron/exon junctions of five analyzed genes. Putative BPS is underlined. PPT is shown by a bracket. Mutant nucleotides are indicated at E+1. (B) RT–PCR of minigenes expressed in HEK293 cells carrying the wild-type (WT) or patient’s (PT) nucleotide. The mutations cause exon skipping in GH1, FECH and EYA1, but not in LPL and HEXA. 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

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

Figure 1: Recapitulation of normal and aberrant splicing of five genes. (A) Nucleotide sequences at the intron/exon junctions of five analyzed genes. Putative BPS is underlined. PPT is shown by a bracket. Mutant nucleotides are indicated at E+1. (B) RT–PCR of minigenes expressed in HEK293 cells carrying the wild-type (WT) or patient’s (PT) nucleotide. The mutations cause exon skipping in GH1, FECH and EYA1, but not in LPL and HEXA. Mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product is shown at the bottom.
Mentions: We first constructed minigenes of GH1, FECH, EYA1, LPL and HEXA, and introduced a previously reported disease-causing mutation at E+1 (Figure 1A). These minigenes successfully recapitulated normal and aberrant splicings: mutations in GH1, FECH and EYA1 caused exon skipping, whereas those in LPL or HEXA did not (Figure 1B).Figure 1.

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