Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

doi:10.1083/jcb.145.3.447

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© The Rockefeller University Press, 0021-9525/1999//447 $5.00
The Journal of Cell Biology, Volume 145, Number 3, , 1999 447-455

Regular Articles

Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

Joanne M. Yeakley^*, Hélène Tronchère^*, James Olesen, Jacqueline A. Dyck^*, Huan-You Wang^*, and Xiang-Dong Fu^*

^* Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La Jolla, California 92093-0651; and Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts 02138

The SR superfamily of splicing factors and regulators is characterizedby arginine/serine (RS)-rich domains, which are extensivelymodified by phosphorylation in cells. In vitro binding studiesrevealed that RS domain–mediated protein interactionscan be differentially affected by phosphorylation. Taking advantageof the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domaininteractions in vivo using the two-hybrid assay. Strikingly,all RS domain–mediated interactions were abolished bySKY1 deletion and were rescuable by yeast or mammalian SR protein–specifickinases, indicating that phosphorylation has a far greater impacton RS domain interactions in vivo than in vitro. To understandthis dramatic effect, we examined the localization of SR proteinsand found that SC35 was shifted to the cytoplasm in sky1 ${Delta}$ yeast,although this phenomenon was not obvious with ASF/SF2, indicatingthat 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 dependon Sky1p to efficiently recognize the LexA binding site ina reporter, suggesting that molecular targeting of RS domain–containingproteins within the nucleus was also affected. Together, theseresults reveal multiple phosphorylation-dependent steps forSR proteins to interact with one another efficiently and specifically,which may ultimately determine the splicing activity and specificityof these factors in mammalian cells.

Key Words: SR proteins • SRPK1 • SRPK2 • Clk • Sty • RS domains

Abbreviations used in this paper: snRNPs, small nuclear ribonucleoprotein particles.

J.M. Yeakley is the recipient of a National Institutes of Health(NIH) postdoctoral fellowship. H. Tronchère was supportedby the Human Frontiers Science Program. J.A. Dyck is fundedby the Damon Runyon-Walter Winchell Cancer Research Foundation.X.-D. Fu is a Leukemia Scholar. This work was supported byan NIH grant to X.-D. Fu.

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