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Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

Yeakley JM, Tronchère H, Olesen J, Dyck JA, Wang HY, Fu XD - J. Cell Biol. (1999)

Bottom Line: Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro.To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation.Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La Jolla, California 92093-0651, USA.

ABSTRACT
The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

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Phosphorylation mediates molecular targeting of RS  domain proteins. Transcriptional repression assays using LexA  fusion proteins performed in wild-type and sky1Δ yeast. Various  bait plasmids were cotransformed with the reporter pJK101 (diagrammed above). Degree of reporter expression is shown as the  percentage of maximal transcription achieved without LexA.
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Figure 5: Phosphorylation mediates molecular targeting of RS domain proteins. Transcriptional repression assays using LexA fusion proteins performed in wild-type and sky1Δ yeast. Various bait plasmids were cotransformed with the reporter pJK101 (diagrammed above). Degree of reporter expression is shown as the percentage of maximal transcription achieved without LexA.

Mentions: To demonstrate that SRPKs play a direct role in assisting RS domain–containing proteins to locate their specific protein or RNA targets, we adapted the following transcriptional repression assay (Golemis and Brent, 1992), which allowed a quantitative measure of phosphorylation-dependent molecular targeting of RS domain proteins in vivo. As diagrammed in Fig. 5, LexA operators were inserted between a GAL1 upstream activating sequence (UAS) and the transcriptional start site for lacZ such that LexA or LexA fusion proteins bind to the operators and repress lacZ expression. In both wild-type and sky1Δ yeast, lacZ expression was constitutive in the absence of LexA, and repressed in its presence. The ability of a control LexA-T3R fusion protein to repress lacZ expression was not affected by the SKY1 deletion. In contrast, the targeting of LexA-SR fusion proteins was dramatically, although not equally, affected by the SKY1 deletion in this assay (Fig. 5). In particular, the LexA-SC35 fusion protein fully repressed lacZ expression in wild-type yeast, but showed little repression in sky1Δ yeast, probably reflecting the significant impact of Sky1p-mediated phosphorylation on the fusion protein at both nuclear localization and intranuclear targeting steps. A similar SKY1-dependent effect was also seen, although less pronounced, with ASF/ SF2, U1-70K, and U2AF65, but was not evident with U2AF35. These observations provide an explanation for the decreased interaction between U2AF65 and U2AF35 in sky1Δ yeast in the two-hybrid assay. The U2AF65 fusion protein expressed from the bait vector in sky1Δ yeast could not efficiently locate its target and activate transcription of the reporter gene, even though targeting of U2AF35 was unaffected by phosphorylation. Based on these results, we conclude that phosphorylation plays a direct role in the molecular targeting of RS domain–containing proteins within the nucleus.


Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

Yeakley JM, Tronchère H, Olesen J, Dyck JA, Wang HY, Fu XD - J. Cell Biol. (1999)

Phosphorylation mediates molecular targeting of RS  domain proteins. Transcriptional repression assays using LexA  fusion proteins performed in wild-type and sky1Δ yeast. Various  bait plasmids were cotransformed with the reporter pJK101 (diagrammed above). Degree of reporter expression is shown as the  percentage of maximal transcription achieved without LexA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2185075&req=5

Figure 5: Phosphorylation mediates molecular targeting of RS domain proteins. Transcriptional repression assays using LexA fusion proteins performed in wild-type and sky1Δ yeast. Various bait plasmids were cotransformed with the reporter pJK101 (diagrammed above). Degree of reporter expression is shown as the percentage of maximal transcription achieved without LexA.
Mentions: To demonstrate that SRPKs play a direct role in assisting RS domain–containing proteins to locate their specific protein or RNA targets, we adapted the following transcriptional repression assay (Golemis and Brent, 1992), which allowed a quantitative measure of phosphorylation-dependent molecular targeting of RS domain proteins in vivo. As diagrammed in Fig. 5, LexA operators were inserted between a GAL1 upstream activating sequence (UAS) and the transcriptional start site for lacZ such that LexA or LexA fusion proteins bind to the operators and repress lacZ expression. In both wild-type and sky1Δ yeast, lacZ expression was constitutive in the absence of LexA, and repressed in its presence. The ability of a control LexA-T3R fusion protein to repress lacZ expression was not affected by the SKY1 deletion. In contrast, the targeting of LexA-SR fusion proteins was dramatically, although not equally, affected by the SKY1 deletion in this assay (Fig. 5). In particular, the LexA-SC35 fusion protein fully repressed lacZ expression in wild-type yeast, but showed little repression in sky1Δ yeast, probably reflecting the significant impact of Sky1p-mediated phosphorylation on the fusion protein at both nuclear localization and intranuclear targeting steps. A similar SKY1-dependent effect was also seen, although less pronounced, with ASF/ SF2, U1-70K, and U2AF65, but was not evident with U2AF35. These observations provide an explanation for the decreased interaction between U2AF65 and U2AF35 in sky1Δ yeast in the two-hybrid assay. The U2AF65 fusion protein expressed from the bait vector in sky1Δ yeast could not efficiently locate its target and activate transcription of the reporter gene, even though targeting of U2AF35 was unaffected by phosphorylation. Based on these results, we conclude that phosphorylation plays a direct role in the molecular targeting of RS domain–containing proteins within the nucleus.

Bottom Line: Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro.To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation.Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La Jolla, California 92093-0651, USA.

ABSTRACT
The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

Show MeSH