UC San Diego

Constitutive and alternative splicing are necessary steps in mRNA processing that contributes to a diverse proteome, but errors in splicing machinery lead to numerous degenerative diseases and cancers. The SR proteins are essential splicing factors that control splice-site selection as well as the transport of processed mRNA for translation. The SR proteins consist of one or two RNA- binding domains and an RS domain rich in arginine-serine repeats. Processive poly-phosphorylation of the RS domain by splicing kinases such as SRPK1, and subsequent dephosphorylation by nuclear phosphatases such as PP1 regulate cellular localization and mRNA processing functions of SR proteins. In this thesis we focused on SRPK1-catalyzed phosphorylation and PP1-catalyzed dephosphorylation of the prototypical SR protein, ASF/SF2. Our overall goals were to establish whether these two enzymes add and remove phosphates in a directional manner and determine specific sequence elements that control initiation and sequential modification of the RS domain of ASF/SF2. A novel LysC digestion phosphomapping technique was developed to observe the order of phosphorylation. We found that SRPK1 phosphorylates ASF/SF2 in a C->N direction preferring to initiate between S221-S225 of ASF/ SF2, but the kinase can adapt to other start sequences if the initiation box is made dysfunctional through mutation. Having established that SRPK1 is a directional kinase with a strong preference for phosphorylation initiation in the center of the RS domain, we then explored structural determinants within the enzyme and substrate that might control this novel mechanism of phosphorylation. Mutagenesis within the RS domain revealed that an uninterrupted RS/SR stretch is necessary for initiation preference. Using the SRPK1:ASF/SF2 complex as a guide for mutagenesis studies, we found that RRM2 (but not RRM1) and N'-RS1 of the RS domain of ASF/SF2 form important and cooperative contacts with SRPK1 that guide directional phosphorylation. Thus, while RS/SR content in the RS domain alone governs regiospecificity, directional phosphorylation is controlled by distal structural elements offered by RRM2 and the docking groove in SRPK1. To analyze how phosphates are removed from the RS domain of ASF/SF2, we studied the dephosphorylation reaction catalyzed by the nuclear protein phosphatase, PP1. PP1 was found to remove phosphates from the RS1 segment of ASF/SF2 in a single kinetic phase suggesting that a common rate- limiting step exists. Deletion analyses indicated that RRM1 protects against dephosphorylation. Through digestion mapping, we found that PP1 dephosphorylates ASF/SF2 in an N->C direction and that removal of RRM1 leads to random dephosphorylation. These data suggest that while RRM2 is important for the directional phosphorylation of ASF/SF2, RRM1 is important for the directional dephosphorylation. Overall, the data suggest that the opposing directional activities of SRPK1 and PP1 may foster the preferential modification of serines in the C-terminal end of RS1 in ASF/SF2, with possible biological relevance