Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1

Abstract

Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore, dsk1؉ gene determines the phenotype of prp2 ؉ overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the doubledeletion mutant. In conjunction with the resemblance of dsk1 ؉ and SRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.Arginine/serine-rich (RS) domain-containing proteins are among the best-characterized non-snRNP proteins participating in pre-mRNA splicing (for reviews, see references 8 and 19). Members of the protein superfamily are involved in constitutive splicing and are specific modulators of alternative splicing (15,19). Mammalian serine/arginine-rich (SR) proteins are featured by one or more RNA recognition motifs at the NH 2 terminus and by an RS domain at the COOH terminus. Other RS domain-containing proteins are relatively less defined with respect to the arrangement of the two structural elements in a protein (8,11,19,35).SR proteins are heavily phosphorylated, predominantly in the RS domain (4,5,12,41). Several kinases have been reported to phosphorylate RS domain-containing splicing factors (5,12,30,39,50,53), including SR protein-specific kinase (SRPK) and Cdc28/Cdc2-like kinase (Clk/Sty). Based on studies in mammalian nuclear extracts, both phosphorylation and dephosphorylation of SR proteins are required for pre-mRNA splicing. Phosphorylation of SR proteins may promote spliceosome assembly by facilitating specific protein interactions while preventing SR proteins from binding randomly to RNA (54). Once a functional spliceosome has formed, dephosphorylation of SR proteins is necessary to allow the transesterification reaction to occur (3, 23). Recently, human type 2C Ser/Thr phosphatase PP2C␥ was reported to be...

Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation

“…Srp2 contains two short SR\RS repeat elements (SR1 and SR2) flanking an arginine-rich region (Figure 1), which is necessary for targeting the protein to the nucleus. The srp2 + gene is essential for cell viability, and its second RBD and the two SR elements are required for its function in i o. SR proteinspecific kinases are conserved in fission yeast [26,29]. Dis1-suppression kinase (Dsk1) protein, originally described as a mitotic regulator [30], displays high activity in phosphorylating S. pombe Srp1 and Srp2 proteins in itro and is a functional homologue of human SRPK1 based on complementation assay in i o.…”

“…Plasmids pET-28bGST, pET-28adsk1 + , pET-28bGST-prp2 + , pET-28asrp1 + , pET-28bGST-srp1 + , pET-28bsrp2 + and pET28bGST-srp2 + have been described previously [26,29]. Recombinant proteins, GST-Srp1, Srp1, GST-Srp2, Srp2, GST-pre-RNA processing 2 (Prp2) and Dsk1, were expressed in the Escherichia coli strain BL21(DE3)pLysS, and the relative amounts of recombinant proteins in lysates were estimated as described previously [26,29].…”

“…Recombinant proteins, GST-Srp1, Srp1, GST-Srp2, Srp2, GST-pre-RNA processing 2 (Prp2) and Dsk1, were expressed in the Escherichia coli strain BL21(DE3)pLysS, and the relative amounts of recombinant proteins in lysates were estimated as described previously [26,29].…”

“…The detailed procedure has been described previously [26]. Briefly, two different bacterial lysates containing a GST-fusion protein and a non-GST-fusion protein were mixed at 23 mC for 30 min to allow complex formation.…”

“…The genome sequence of S. pombe reveals that nearly 25 % of its genes contain multiple introns [22,23], and some of them have been shown to undergo alternative splicing [24,25]. Moreover, SR-related proteins (Srps) and SRprotein-specific kinases exist in S. pombe [21,26]. Thus S. pombe has the potential to be a good model system for studying the function of SR proteins in splicing regulation.…”