Abstract
BackgroundThe U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells.ResultsOverexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1.ConclusionThe present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.
Highlights
The U2 small nuclear ribonucleoprotein particle component SF3b1/ SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis
The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing
In order to further characterise SF3b1 as a substrate of DYRK1A, we performed a kinetic analysis of the phosphorylation of His6-SF3b1304– 493, the fusion protein produced from the library clone, by GST-DYRK1A-∆C
Summary
The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/ SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. The excision of introns from pre-mRNA is catalysed by the spliceosome, a macromolecular machine consisting of five small nuclear ribonucleoprotein particles (snRNPs) and a large number of non-snRNP proteins [1]. The spliceosome dissociates into its snRNP subunits, which take part in ensuing rounds of splicing. The best studied targets for phosphorylation are members of the SR family of splicing factors, which contain domains rich in Arg/Ser dipeptides [4]. Several kinases phosphorylate these RS domains and modulate interaction of SR proteins with other proteins during spliceosome assembly [5]. Phosphorylation affects the intranuclear distribution of splicing factors and alternative splice site selection [6,7,8,9,10]
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