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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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RT–PCR of HEK293 cells transfected with minigenes carrying artificially extended or disrupted PPT’s. All the constructs harbor a mutation at E+1. The top construct of each gene represents the patient’s sequence. Only the nucleotide sequences of the 3′-end of an intron are indicated. The longest stretches of the polypyrimidines are shown in bold. Underlines indicate putative BPS’s. The rightmost column shows the mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product.
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Figure 4: RT–PCR of HEK293 cells transfected with minigenes carrying artificially extended or disrupted PPT’s. All the constructs harbor a mutation at E+1. The top construct of each gene represents the patient’s sequence. Only the nucleotide sequences of the 3′-end of an intron are indicated. The longest stretches of the polypyrimidines are shown in bold. Underlines indicate putative BPS’s. The rightmost column shows the mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product.

Mentions: In an effort to delineate effects of the PPT sequences on the splicing consequence of a mutation at E+1, we introduced a series of mutations into the PPT in the presence of the mutation at E+1. Extensions of the polypyrimidine stretch ameliorated aberrant splicing in GH1, FECH and EYA1. Conversely, truncations or disruptions of the polypyrimidine stretch caused exon skipping in LPL and HEXA (Figure 4).Figure 4.


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)

RT–PCR of HEK293 cells transfected with minigenes carrying artificially extended or disrupted PPT’s. All the constructs harbor a mutation at E+1. The top construct of each gene represents the patient’s sequence. Only the nucleotide sequences of the 3′-end of an intron are indicated. The longest stretches of the polypyrimidines are shown in bold. Underlines indicate putative BPS’s. The rightmost column shows the mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: RT–PCR of HEK293 cells transfected with minigenes carrying artificially extended or disrupted PPT’s. All the constructs harbor a mutation at E+1. The top construct of each gene represents the patient’s sequence. Only the nucleotide sequences of the 3′-end of an intron are indicated. The longest stretches of the polypyrimidines are shown in bold. Underlines indicate putative BPS’s. The rightmost column shows the mean and SD of three independent experiments of the densitometric ratios of the exon-skipped product.
Mentions: In an effort to delineate effects of the PPT sequences on the splicing consequence of a mutation at E+1, we introduced a series of mutations into the PPT in the presence of the mutation at E+1. Extensions of the polypyrimidine stretch ameliorated aberrant splicing in GH1, FECH and EYA1. Conversely, truncations or disruptions of the polypyrimidine stretch caused exon skipping in LPL and HEXA (Figure 4).Figure 4.

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