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

The SR superfamily of splicing factors and regulators is characterized by a rginine/serine (RS)-rich domains, which are extensively modified by phospho rylation in cells. In vitro binding studies revealed that RS domain-mediate d protein interactions can be differentially affected by phosphorylation, T aking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in viv o using the two-hybrid assay. Strikingly, all RS domain-mediated interactio ns were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far gre ater 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 sky1 Delta 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 tra nscriptional repression assay, we further showed that most LexA-SR fusion p roteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing prot eins within the nucleus was also affected. Together, these results reveal m ultiple phosphorylation-dependent steps for SR proteins to interact with on e another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.