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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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Sam68 and pre-mRNA occupancy by U2AF.(A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to Figure 4. (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.
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pone-0001418-g005: Sam68 and pre-mRNA occupancy by U2AF.(A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to Figure 4. (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.

Mentions: Given the interaction of Sam68 and U2AF65, binding of Sam68 might stabilize association of U2AF with pre-mRNA. If this were true, the rapid release from RNA of Sam68 upon phorbol-ester treatment of the cells, should result in decreased pre-mRNA occupancy by U2AF65 in vivo. To test this prediction, we performed RNP immunoprecipitation experiments using an anti-U2AF65 antibody. We could specifically precipitate RNA fragments containing the CD44 v5 exon and the upstream intron sequence (Figure 5A, lanes 1 and 2). Furthermore, phorbol-ester treatment of the cells for ten minutes led to a marked reduction of the fragments detectable in the U2AF immunoprecipitate (Figure 5A, compare lanes 2 and 3). This finding supports the idea that RNA-bound Sam68 can act as a stabilizing factor for U2AF binding. To test this idea further, we examined if forced expression of Sam68 can increase the occupancy of this pre-mRNA region by U2AF65. Figure 5C shows that co-transfection of a Sam68 expression vector led to an increase in the RNA fragments precipitated with the anti-U2AF65 antibody when compared to control-vector transfected cells (lanes 2 and 3). Together with the GST pull-down and co-immunoprecipitation data, these findings point to Sam68 as a factor that can stabilize U2AF binding by protein-protein interaction in vivo. Moreover, they suggest that Ras-signaling-induced phosphorylation of Sam68 interferes with binding of Sam68 to RNA, and may thus affect splice site occupancy by U2AF65.


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

Tisserant A, König H - PLoS ONE (2008)

Sam68 and pre-mRNA occupancy by U2AF.(A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to Figure 4. (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169300&req=5

pone-0001418-g005: Sam68 and pre-mRNA occupancy by U2AF.(A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to Figure 4. (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.
Mentions: Given the interaction of Sam68 and U2AF65, binding of Sam68 might stabilize association of U2AF with pre-mRNA. If this were true, the rapid release from RNA of Sam68 upon phorbol-ester treatment of the cells, should result in decreased pre-mRNA occupancy by U2AF65 in vivo. To test this prediction, we performed RNP immunoprecipitation experiments using an anti-U2AF65 antibody. We could specifically precipitate RNA fragments containing the CD44 v5 exon and the upstream intron sequence (Figure 5A, lanes 1 and 2). Furthermore, phorbol-ester treatment of the cells for ten minutes led to a marked reduction of the fragments detectable in the U2AF immunoprecipitate (Figure 5A, compare lanes 2 and 3). This finding supports the idea that RNA-bound Sam68 can act as a stabilizing factor for U2AF binding. To test this idea further, we examined if forced expression of Sam68 can increase the occupancy of this pre-mRNA region by U2AF65. Figure 5C shows that co-transfection of a Sam68 expression vector led to an increase in the RNA fragments precipitated with the anti-U2AF65 antibody when compared to control-vector transfected cells (lanes 2 and 3). Together with the GST pull-down and co-immunoprecipitation data, these findings point to Sam68 as a factor that can stabilize U2AF binding by protein-protein interaction in vivo. Moreover, they suggest that Ras-signaling-induced phosphorylation of Sam68 interferes with binding of Sam68 to RNA, and may thus affect splice site occupancy by U2AF65.

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