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Characterization of the RRM2 domain of splicing factor ASF /SF2


Splicing of pre-mRNA transcripts in eukayrotes is mediated by a complex macromolecule consisting of ribonucleoproteins and several accessory protein factors. The SR protein is one family of such factor. They are characterized by the presence of at least one N-terminal RNA recognition motif (RRM) and a C-terminal domain rich in arginine-serine dipeptides (RS domain), which are extensively phosphorylated by SRPK and Clk/Sty kinases. It has been shown that SRPK1 bind ASF/SF2 with unusual high affinity and processively phosphorylates half of the RS domain. Our lab used structural studies to investigate this interaction and phosphorylation mechanism. Crystal structure of SRPK1:ASF/SF2 complex revealed bipartite interactions: RRM2 of ASF/SF2 contacts both lobes of the kinase and the RS domain binds to a docking groove in the kinase. We have shown that this docking interaction is critical for phosphorylation and subcellular localization of ASF/SF2. My thesis works attempted to investigate the contribution of RRM2 to the high affinity interaction. My results showed RRM2 alone cannot bind SRPK1. Only after the RS domain has docked in the kinase groove, we saw enhancement in interaction with RRM2. We believe that the interaction at the interface of RRM2:SRPK1 is not affinity -based but is an allosteric-induced mode of interaction. This is consistent with a recent study showing that RS domain alone can initiate processive phosphorylation, but interaction with the RRM modules is required to maintain processitivity

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