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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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RNA-EMSA. (A) Sequences of wild-type (WT) and mutant (PT) RNA probes of FECH and LPL employed for RNA-EMSA. (B) RNA-EMSA of wild-type and mutant FECH and LPL with increasing amounts of U2AF65 with or without U2AF35. His-tagged U2AF65 and U2AF35 are expressed in Sf9 cells and are purified. Wild-type FECH requires U2AF35 to bind to U2AF65, whereas wild-type LPL does not require U2AF35. A mutation at E+1 abrogates binding of U2AF65 in FECH but not in LPL. Concentrations of U2AF35 are 5, 10 and 20 ng/µl; and those of U2AF65 are 10, 20 and 40 ng/µl. Numbers at the bottom indicate intensities of the retarded fragments in arbitrary units.
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Figure 3: RNA-EMSA. (A) Sequences of wild-type (WT) and mutant (PT) RNA probes of FECH and LPL employed for RNA-EMSA. (B) RNA-EMSA of wild-type and mutant FECH and LPL with increasing amounts of U2AF65 with or without U2AF35. His-tagged U2AF65 and U2AF35 are expressed in Sf9 cells and are purified. Wild-type FECH requires U2AF35 to bind to U2AF65, whereas wild-type LPL does not require U2AF35. A mutation at E+1 abrogates binding of U2AF65 in FECH but not in LPL. Concentrations of U2AF35 are 5, 10 and 20 ng/µl; and those of U2AF65 are 10, 20 and 40 ng/µl. Numbers at the bottom indicate intensities of the retarded fragments in arbitrary units.

Mentions: To further prove that U2AF35 is required for pre-mRNA splicing, we employed an electrophoretic mobility shift assay (EMSA) using wild-type and mutant RNA substrates of FECH and LPL (Figure 3A). His-tagged U2AF35 and U2AF65 were expressed using bacluovirus and were purified under denatured and native conditions, respectively. Denatured U2AF35 was refolded before RNA-EMSA. As expected, U2AF65 failed to bind to the wild-type FECH in the absence of U2AF35, and addition of U2AF35 gained its binding. For the mutant FECH, neither U2AF65 alone nor addition of both U2AFs showed binding of U2AFs. On the other hand, the wild-type LPL did not require U2AF35 to bind to U2AF65. Addition of U2AF35 did not substantially increased binding of U2AF65. These bindings were not affected by the mutation at E+1 of LPL (Figure 3B).Figure 3.


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)

RNA-EMSA. (A) Sequences of wild-type (WT) and mutant (PT) RNA probes of FECH and LPL employed for RNA-EMSA. (B) RNA-EMSA of wild-type and mutant FECH and LPL with increasing amounts of U2AF65 with or without U2AF35. His-tagged U2AF65 and U2AF35 are expressed in Sf9 cells and are purified. Wild-type FECH requires U2AF35 to bind to U2AF65, whereas wild-type LPL does not require U2AF35. A mutation at E+1 abrogates binding of U2AF65 in FECH but not in LPL. Concentrations of U2AF35 are 5, 10 and 20 ng/µl; and those of U2AF65 are 10, 20 and 40 ng/µl. Numbers at the bottom indicate intensities of the retarded fragments in arbitrary units.
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Figure 3: RNA-EMSA. (A) Sequences of wild-type (WT) and mutant (PT) RNA probes of FECH and LPL employed for RNA-EMSA. (B) RNA-EMSA of wild-type and mutant FECH and LPL with increasing amounts of U2AF65 with or without U2AF35. His-tagged U2AF65 and U2AF35 are expressed in Sf9 cells and are purified. Wild-type FECH requires U2AF35 to bind to U2AF65, whereas wild-type LPL does not require U2AF35. A mutation at E+1 abrogates binding of U2AF65 in FECH but not in LPL. Concentrations of U2AF35 are 5, 10 and 20 ng/µl; and those of U2AF65 are 10, 20 and 40 ng/µl. Numbers at the bottom indicate intensities of the retarded fragments in arbitrary units.
Mentions: To further prove that U2AF35 is required for pre-mRNA splicing, we employed an electrophoretic mobility shift assay (EMSA) using wild-type and mutant RNA substrates of FECH and LPL (Figure 3A). His-tagged U2AF35 and U2AF65 were expressed using bacluovirus and were purified under denatured and native conditions, respectively. Denatured U2AF35 was refolded before RNA-EMSA. As expected, U2AF65 failed to bind to the wild-type FECH in the absence of U2AF35, and addition of U2AF35 gained its binding. For the mutant FECH, neither U2AF65 alone nor addition of both U2AFs showed binding of U2AFs. On the other hand, the wild-type LPL did not require U2AF35 to bind to U2AF65. Addition of U2AF35 did not substantially increased binding of U2AF65. These bindings were not affected by the mutation at E+1 of LPL (Figure 3B).Figure 3.

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