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Signal-regulated Pre-mRNA occupancy by the general splicing factor U2AF.

Tisserant A, König H - PLoS ONE (2008)

Bottom Line: We show here that U2AF interacts with the signal-dependent splice regulator Sam68 and that forced expression of Sam68 results in enhanced binding of the U2AF65 subunit to an alternatively spliced pre-mRNA sequence in vivo.Conversely, the rapid signal-induced and phosphorylation-dependent interference with Sam68 binding to RNA was accompanied by reduced pre-mRNA occupancy of U2AF in vivo.Our data suggest that Sam68 can affect splice site occupancy by U2AF in signal-dependent splicing.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe GmbH, Institut für Toxikologie und Genetik, Karlsruhe, Germany.

ABSTRACT
Alternative splicing of transcripts in a signal-dependent manner has emerged as an important concept to ensure appropriate expression of splice variants under different conditions. Binding of the general splicing factor U2AF to splice sites preceding alternatively spliced exons has been suggested to be an important step for splice site recognition. For splicing to proceed, U2AF has to be replaced by other factors. We show here that U2AF interacts with the signal-dependent splice regulator Sam68 and that forced expression of Sam68 results in enhanced binding of the U2AF65 subunit to an alternatively spliced pre-mRNA sequence in vivo. Conversely, the rapid signal-induced and phosphorylation-dependent interference with Sam68 binding to RNA was accompanied by reduced pre-mRNA occupancy of U2AF in vivo. Our data suggest that Sam68 can affect splice site occupancy by U2AF in signal-dependent splicing. We propose that the induced release of U2AF from pre-mRNA provides a regulatory step to control alternative splicing.

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Intronic and exonic Sam68 binding sites affect CD44 v5 exon inclusion.(A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes [15]. They were mutated for either the exonic (L/CC mutant [19]), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml−1) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.
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pone-0001418-g002: Intronic and exonic Sam68 binding sites affect CD44 v5 exon inclusion.(A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes [15]. They were mutated for either the exonic (L/CC mutant [19]), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml−1) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.

Mentions: A Sam68-binding sequence in CD44 exon v5 was shown to determine the overall level of exon inclusion [19]. We found a second site corresponding to the Sam68 consensus binding sequence [22] in the intron preceding the v5 exon, immediately upstream of the putative branch point sequence (Figure 2A). To test whether this second site can indeed bind Sam68, we performed electrophoretic mobility shift assays (EMSA) involving recombinant Sam68 and RNA oligonucleotides comprising this site or a point-mutated version of it (A/C mutant; see Figure 2A). This mutation should interfere with Sam68 binding [22]. As shown in Figure 2B, the RNA oligonucleotide comprising the wild-type sequence bound Sam68 in a dose-dependent manner (lanes 1–4), whereas binding to the A/C-mutated version was severely compromised (lanes 5–8), indicating that Sam68 binds to the oligonucleotide depending on this sequence. To test the functional relevance of this intronic Sam68 binding-element, we introduced the A/C mutation into a luciferase-based splice-reporter minigene carrying CD44 exon v5 [15] and transfected it into LB17 mouse lymphoma cells. In these cells inclusion of the v5 exon in mRNA can be induced by Ras-pathway activation upon phorbol-ester treatment [15], [23]. Similar to the mutation of the exonic Sam68 binding site [19], the A/C mutation of the intronic Sam68 reduced the overall level of v5 exon inclusion, whereas induction of exon inclusion by phorbol ester treatment was still functional (Figure 2C). The same was true for a construct in which both Sam68 binding sites were mutated (Figure 2C). These results suggest that binding of Sam68 to these elements in the CD44 pre-mRNA determines the overall level of v5 exon inclusion in mRNA. Signal-mediated induction of splicing, although dependent on Sam68 [19], seems however not to depend on these sites. Could there still be a link between Sam68 bound to these RNA elements and splicing induction? To address this question, we initially performed electrophoretic mobility shift assays (EMSA) employing RNA oligonucleotides comprising either of the two Sam68 binding elements and different amounts of either non-phosphorylated Sam68 or of Sam68 thiophosphorylated by ERK in vitro (Figure 3). The stable thiophosphorylation results in the same shift in molecular weight (see Figure 3A) as compared to normal phophorylation and is active in inducing v5 exon splicing in vitro [19]. So phosphorylated Sam68 consistently shifted less radioactively labeled oligonucleotide as compared to non-phosphorylated Sam68, both with the exonic element (Figure 3B, lanes 2–5) and the intronic element probe (Figure 3B, lanes 7–10). This result suggests that phosphorylation of Sam68 by ERK interferes with the ability of Sam68 to bind its cognate RNA binding elements in vitro.


Signal-regulated Pre-mRNA occupancy by the general splicing factor U2AF.

Tisserant A, König H - PLoS ONE (2008)

Intronic and exonic Sam68 binding sites affect CD44 v5 exon inclusion.(A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes [15]. They were mutated for either the exonic (L/CC mutant [19]), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml−1) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169300&req=5

pone-0001418-g002: Intronic and exonic Sam68 binding sites affect CD44 v5 exon inclusion.(A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes [15]. They were mutated for either the exonic (L/CC mutant [19]), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml−1) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.
Mentions: A Sam68-binding sequence in CD44 exon v5 was shown to determine the overall level of exon inclusion [19]. We found a second site corresponding to the Sam68 consensus binding sequence [22] in the intron preceding the v5 exon, immediately upstream of the putative branch point sequence (Figure 2A). To test whether this second site can indeed bind Sam68, we performed electrophoretic mobility shift assays (EMSA) involving recombinant Sam68 and RNA oligonucleotides comprising this site or a point-mutated version of it (A/C mutant; see Figure 2A). This mutation should interfere with Sam68 binding [22]. As shown in Figure 2B, the RNA oligonucleotide comprising the wild-type sequence bound Sam68 in a dose-dependent manner (lanes 1–4), whereas binding to the A/C-mutated version was severely compromised (lanes 5–8), indicating that Sam68 binds to the oligonucleotide depending on this sequence. To test the functional relevance of this intronic Sam68 binding-element, we introduced the A/C mutation into a luciferase-based splice-reporter minigene carrying CD44 exon v5 [15] and transfected it into LB17 mouse lymphoma cells. In these cells inclusion of the v5 exon in mRNA can be induced by Ras-pathway activation upon phorbol-ester treatment [15], [23]. Similar to the mutation of the exonic Sam68 binding site [19], the A/C mutation of the intronic Sam68 reduced the overall level of v5 exon inclusion, whereas induction of exon inclusion by phorbol ester treatment was still functional (Figure 2C). The same was true for a construct in which both Sam68 binding sites were mutated (Figure 2C). These results suggest that binding of Sam68 to these elements in the CD44 pre-mRNA determines the overall level of v5 exon inclusion in mRNA. Signal-mediated induction of splicing, although dependent on Sam68 [19], seems however not to depend on these sites. Could there still be a link between Sam68 bound to these RNA elements and splicing induction? To address this question, we initially performed electrophoretic mobility shift assays (EMSA) employing RNA oligonucleotides comprising either of the two Sam68 binding elements and different amounts of either non-phosphorylated Sam68 or of Sam68 thiophosphorylated by ERK in vitro (Figure 3). The stable thiophosphorylation results in the same shift in molecular weight (see Figure 3A) as compared to normal phophorylation and is active in inducing v5 exon splicing in vitro [19]. So phosphorylated Sam68 consistently shifted less radioactively labeled oligonucleotide as compared to non-phosphorylated Sam68, both with the exonic element (Figure 3B, lanes 2–5) and the intronic element probe (Figure 3B, lanes 7–10). This result suggests that phosphorylation of Sam68 by ERK interferes with the ability of Sam68 to bind its cognate RNA binding elements in vitro.

Bottom Line: We show here that U2AF interacts with the signal-dependent splice regulator Sam68 and that forced expression of Sam68 results in enhanced binding of the U2AF65 subunit to an alternatively spliced pre-mRNA sequence in vivo.Conversely, the rapid signal-induced and phosphorylation-dependent interference with Sam68 binding to RNA was accompanied by reduced pre-mRNA occupancy of U2AF in vivo.Our data suggest that Sam68 can affect splice site occupancy by U2AF in signal-dependent splicing.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe GmbH, Institut für Toxikologie und Genetik, Karlsruhe, Germany.

ABSTRACT
Alternative splicing of transcripts in a signal-dependent manner has emerged as an important concept to ensure appropriate expression of splice variants under different conditions. Binding of the general splicing factor U2AF to splice sites preceding alternatively spliced exons has been suggested to be an important step for splice site recognition. For splicing to proceed, U2AF has to be replaced by other factors. We show here that U2AF interacts with the signal-dependent splice regulator Sam68 and that forced expression of Sam68 results in enhanced binding of the U2AF65 subunit to an alternatively spliced pre-mRNA sequence in vivo. Conversely, the rapid signal-induced and phosphorylation-dependent interference with Sam68 binding to RNA was accompanied by reduced pre-mRNA occupancy of U2AF in vivo. Our data suggest that Sam68 can affect splice site occupancy by U2AF in signal-dependent splicing. We propose that the induced release of U2AF from pre-mRNA provides a regulatory step to control alternative splicing.

Show MeSH
Related in: MedlinePlus