Limits...
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.

Show MeSH

Related in: MedlinePlus

Sam68 interacts with U2AF in vitro and in vivo.(A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169300&req=5

pone-0001418-g001: Sam68 interacts with U2AF in vitro and in vivo.(A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).

Mentions: To test the possibility that Sam68 could affect U2AF binding by protein-protein interaction, we initially tested whether the two proteins can interact in vitro. Therefore, we performed pull-down experiments with bacterially expressed Glutathione S-transferase (GST) fusion proteins of U2AF65 and U2AF35, respectively, and recombinant His-tagged Sam68. As shown in Figure 1A by immunoblotting with an anti-Sam68 antibody, both subunits of U2AF precipitated Sam68 in a dose-dependent manner (lanes 1–4). This was not the case when we performed the assays with the GST portion alone (lanes 5–6), suggesting U2AF-dependent interaction. To investigate whether Sam68 could be complexed with U2AF in vivo, we performed co-immunoprecipitations with an anti-Sam68 antibody and lysates from LB17 mouse lymphoma cells. Immunoblotting with an anti-U2AF65 antibody showed that the anti-Sam68 precipitate contained U2AF65 (Figure 1B, compare lanes 2 and 3). Precipitates from the control antibody did not show the U2AF65 band (Figure 1B, lane 1), indicating Sam68-dependent precipitation of the protein. Treatment of the lysates prior and during precipitation by RNase A did not interfere with co-precipitation of U2AF65 (Figure 1C, compare lanes 2 and 3), pointing to the association of the proteins independent of RNA bridging. Due to the poor sensitivity of the U2AF35 antibody available, we could not test the presence of U2AF35 in the precipitates. Taken together these data suggest that Sam68 can directly interact with U2AF proteins and that Sam68 is associated with U2AF65 in vivo.


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

Tisserant A, König H - PLoS ONE (2008)

Sam68 interacts with U2AF in vitro and in vivo.(A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001418-g001: Sam68 interacts with U2AF in vitro and in vivo.(A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).
Mentions: To test the possibility that Sam68 could affect U2AF binding by protein-protein interaction, we initially tested whether the two proteins can interact in vitro. Therefore, we performed pull-down experiments with bacterially expressed Glutathione S-transferase (GST) fusion proteins of U2AF65 and U2AF35, respectively, and recombinant His-tagged Sam68. As shown in Figure 1A by immunoblotting with an anti-Sam68 antibody, both subunits of U2AF precipitated Sam68 in a dose-dependent manner (lanes 1–4). This was not the case when we performed the assays with the GST portion alone (lanes 5–6), suggesting U2AF-dependent interaction. To investigate whether Sam68 could be complexed with U2AF in vivo, we performed co-immunoprecipitations with an anti-Sam68 antibody and lysates from LB17 mouse lymphoma cells. Immunoblotting with an anti-U2AF65 antibody showed that the anti-Sam68 precipitate contained U2AF65 (Figure 1B, compare lanes 2 and 3). Precipitates from the control antibody did not show the U2AF65 band (Figure 1B, lane 1), indicating Sam68-dependent precipitation of the protein. Treatment of the lysates prior and during precipitation by RNase A did not interfere with co-precipitation of U2AF65 (Figure 1C, compare lanes 2 and 3), pointing to the association of the proteins independent of RNA bridging. Due to the poor sensitivity of the U2AF35 antibody available, we could not test the presence of U2AF35 in the precipitates. Taken together these data suggest that Sam68 can directly interact with U2AF proteins and that Sam68 is associated with U2AF65 in vivo.

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