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]. Spliceosome assembly proceeds via the step-wise recruitment of U1 snRNP, U2 snRNP, and U4/U6·U5 tri-snRNP on a pre-mRNA as well as multiple rearrangements between the spliceosomal components [1]. After splicing catalysis, the spliceosome dissociates into its snRNP subunits, which take part in ensuing rounds of splicing.
Both spliceosome assembly and splicing catalysis is regulated by reversible protein phosphorylation [1-3]. 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]. In addition, phosphorylation affects the intranuclear distribution of splicing factors and alternative splice site selection [6-10].
The only non-SR component of the spliceosome known to be phosphorylated during splicing catalysis is SF3b1 (also called SAP155 or SF3b155), one of the subunits of the U2 snRNP-associated complex SF3b [3,11]. SF3b1 is positioned at the spliceosome catalytic center and contacts pre-mRNA on both sides of the branch site [12]. Phosphorylation of SF3b1 appears to be functionally important in the basic splicing reaction as it is detected only in functional spliceosomes and occurs concomitant with splicing catalysis [3]. The N-terminal part of SF3b1 contains abundant Thr-Pro dipeptides motifs which are potential phosphorylation sites of proline-directed kinases like the cyclin-dependent kinases (CDK). Indeed, cyclin E/CDK2 has been shown to phosphorylate SF3b1 in vitro and to be associated with the U2 snRNP complex in vivo [11].
We have recently identified several splicing factors, including SF3b1, as substrates of the protein kinase DYRK1A [13]. DYRK1A is a nuclear protein kinase that has been localised to the splicing factor compartment [14]. Furthermore, we have previously characterised DYRK1A as a kinase that targets serine/threonine followed by a proline residue [15].
Here we report that DYRK1A efficiently phosphorylates SF3b1 within the TP-rich domain at several sites that are also phosphorylated by endogenous kinases in COS-7 cells. One of these sites, Thr434, was identified as the residue predominantly phosphorylated by DYRK1A in vitro and as a major phosphorylation site of SF3b1 in vivo.
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