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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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Effects of down-regulation of U2AF35 on pre-mRNA splicing. (A) Western blots demonstrating that U2AF35-siRNA efficiently knocks down U2AF35 but not U2AF65 or PTBP1. (B) Down-regulation of U2AF35 facilitates exon skipping in wild-type GH1 and FECH, but not in wild-type EYA1, LPL and HEXA. (C) Introduction of an siRNA-resistant p3XFLAG-U2AF35 encoding 3× FLAG fused with U2AF35 is visualized by immunoblots against FLAG and U2AF35. (D) Exon skipping facilitated by U2AF35-siRNA is partially rescued by introduction of the siRNA-resistant p3XFLAG-U2AF35.
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Figure 2: Effects of down-regulation of U2AF35 on pre-mRNA splicing. (A) Western blots demonstrating that U2AF35-siRNA efficiently knocks down U2AF35 but not U2AF65 or PTBP1. (B) Down-regulation of U2AF35 facilitates exon skipping in wild-type GH1 and FECH, but not in wild-type EYA1, LPL and HEXA. (C) Introduction of an siRNA-resistant p3XFLAG-U2AF35 encoding 3× FLAG fused with U2AF35 is visualized by immunoblots against FLAG and U2AF35. (D) Exon skipping facilitated by U2AF35-siRNA is partially rescued by introduction of the siRNA-resistant p3XFLAG-U2AF35.

Mentions: We predicted that a mutation at E+1 should disrupt binding of U2AF35. We thus hypothesized that GH1, FECH and EYA1 require binding of U2AF35 for the assembly of spliceosome, whereas LPL and HEXA do not require it. To prove this hypothesis, we first knocked down U2AF35 and analyzed its effect on the wild-type minigenes. We achieved an efficient down-regulation of U2AF35 in HEK293 cells (Figure 2A). We also confirmed that the U2AF35-siRNA had no effect on the expression level of U2AF65. As expected, the down-regulation of U2AF35 increased exon skipping of GH1 and FECH (Figure 2B) but not to the levels of the mutant constructs (Figure 1B). Again, as expected, we observed no effect on LPL and HEXA. Unexpectedly, however, EYA1 demonstrated no response to the down-regulation of U2AF35. Less efficient effects of U2AF35-siRNA on GH1, FECH and EYA1 (Figure 2B) compared to the mutant constructs (Figure 1B) were likely because the mutation abolished binding of U2AF35 in all the cells, whereas substantial numbers of cells failed to incorporate U2AF35-siRNA and gave rise to normally spliced products.Figure 2.


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)

Effects of down-regulation of U2AF35 on pre-mRNA splicing. (A) Western blots demonstrating that U2AF35-siRNA efficiently knocks down U2AF35 but not U2AF65 or PTBP1. (B) Down-regulation of U2AF35 facilitates exon skipping in wild-type GH1 and FECH, but not in wild-type EYA1, LPL and HEXA. (C) Introduction of an siRNA-resistant p3XFLAG-U2AF35 encoding 3× FLAG fused with U2AF35 is visualized by immunoblots against FLAG and U2AF35. (D) Exon skipping facilitated by U2AF35-siRNA is partially rescued by introduction of the siRNA-resistant p3XFLAG-U2AF35.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Effects of down-regulation of U2AF35 on pre-mRNA splicing. (A) Western blots demonstrating that U2AF35-siRNA efficiently knocks down U2AF35 but not U2AF65 or PTBP1. (B) Down-regulation of U2AF35 facilitates exon skipping in wild-type GH1 and FECH, but not in wild-type EYA1, LPL and HEXA. (C) Introduction of an siRNA-resistant p3XFLAG-U2AF35 encoding 3× FLAG fused with U2AF35 is visualized by immunoblots against FLAG and U2AF35. (D) Exon skipping facilitated by U2AF35-siRNA is partially rescued by introduction of the siRNA-resistant p3XFLAG-U2AF35.
Mentions: We predicted that a mutation at E+1 should disrupt binding of U2AF35. We thus hypothesized that GH1, FECH and EYA1 require binding of U2AF35 for the assembly of spliceosome, whereas LPL and HEXA do not require it. To prove this hypothesis, we first knocked down U2AF35 and analyzed its effect on the wild-type minigenes. We achieved an efficient down-regulation of U2AF35 in HEK293 cells (Figure 2A). We also confirmed that the U2AF35-siRNA had no effect on the expression level of U2AF65. As expected, the down-regulation of U2AF35 increased exon skipping of GH1 and FECH (Figure 2B) but not to the levels of the mutant constructs (Figure 1B). Again, as expected, we observed no effect on LPL and HEXA. Unexpectedly, however, EYA1 demonstrated no response to the down-regulation of U2AF35. Less efficient effects of U2AF35-siRNA on GH1, FECH and EYA1 (Figure 2B) compared to the mutant constructs (Figure 1B) were likely because the mutation abolished binding of U2AF35 in all the cells, whereas substantial numbers of cells failed to incorporate U2AF35-siRNA and gave rise to normally spliced products.Figure 2.

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