Phosphorylation Of Splicing Factor SRp55 By Dyrk1A

Alternative splicing, a post-transcriptional modification, is carried out in splicesome to produce altered mRNA from pre-mRNA. SR proteins are important splicing factors. They contain one or two RNA recognition motif(s) (RRMs), together with a carboxyl terminal domain containing numerous arginine-serine amino acid pairs (RS domain). The RS domain mediates protein-protein interactions and whereas the RRMs are responsible for specificity of substrates. Up to date, 10 SR proteins have been found. They all are phospho-proteins and phosphorylation tightly regulates their function and intracellular localization. SRp55, one of the SR proteins, has been shown to participate the alternative splicing of some genes including MAPT (microtubule-associated protein tau). It has many potential phosphorylation sites and its function in splicing is regulated by phosphorylation.Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A), the mammalian orthologue of Drosophila minibrain kinase (Mnb), encodes a proline/arginine-directed serine/threonine kinase. It locates in Down syndrome (DS) critical region and contributes to many phenotypes of DS. The level and the activity of Dyrk1A are increased by 50 % in DS brain due to trisomy 21. Increased studies have shown that dysregulation of alternative splicing may contribute its pathogenesis. In present study, we studied the interaction and phosphorylation of SRp55 with or by Dyrk1A. By using GST-pull down assay, co-immunoprecipitation and co-localization, we found that SRp55 interacted with Dyrk1A and the interaction was mediated by RS2 sub-domain. Dyrk1A phosphorylated in vitro SRp55 significantly in enzyme concentration dependent manner. The phosphorylation sites were mainly located within RS1 and pro-rich sub-domains of SRp55 RS domain. Overexpression of Dyrk1A induced a translocation of SRp55 to speckles. Deletion of RS2 sub-domain, which mediates the SRp55 interaction with Dyrk1A,prevented the SRp55 enrichment in the speckles. To study the role of Dyrk1A in SRp55-mediated alternative splicing, we transfected mini-tau gene (pCI/SI9/SI10) into HeLa, COS7, SH-SY5Y and HEK293-T cells. We found that SRp55 enhanced tau exon 10 inclusion and Dyrk1A modulated the enhancement of exon 10 inclusion by SRp55, which suggest that in addition to inducing the translocation of SRp55, phosphorylation by Dyrk1A may also regulate the activity of SRp55 in alternative splicing.Conclusion:1. Dyrk1A phosphorylates SRp55 in vitro on sites within RS1 and pro-rich sub-domain of SRp55.2. SRp55 interacts with Dyrk1A and RS2 sub-domain mediated the interaction.3. Over-expression of Dyrk1A induces the translocation of SRp55 to speckles. RS2-deleted SRp55 has a weaker ability to accumulate in speckles.4. Dyrk1A regulates SRp55-mediated alternative splicing such as tau exon 10.