Many human diseases result from irregularities in mRNA splicing, a process catalyzed by the spliceosome. As vital components of the spliceosome, SR proteins establish splice sites in the precursor mRNA. They contain RNA recognition motifs (RRMs) and an RS domain that is polyphosphorylated by SRPK1. The latter modifications control SR protein function, thereby regulating the splicing of human genes. Recent kinetic and crystallographic studies indicate that SRPK1 phosphorylates the RS domain of the SR protein, ASF/SF2, using a mechanism that is directional, regiospecific, and processive. As the interaction of ASF/SF2 with SRPK1 is necessary for splicing activity, understanding how SRPK1 recognizes the SR protein subdomains is important for gaining a better understanding of mRNA splicing. In this proposal, the mechanism of SRPK1-ASF/SF2 complex formation will be investigated using a variety of kinetic and spectrometric techniques. As the domains of ASF/SF2 make numerous contacts with SRPK1, guide regiospecific phosphorylation and link to splicing activity, the binding order of these domains will be studied using fluorescence and autoradiographic methods under equilibrium and transient-state conditions. A new fluorescence assay for SR protein phosphorylation has been developed and will be explored to address how RRM and RS domain binding are structurally linked. Recent data show that a large insert domain in SRPK1 functions as a cytoplasmic anchor by interacting with chaperone proteins. It also acts as an allosteric regulator that promotes cross-talk between a docking groove in SRPK1 that binds the RS domain and a region that interacts with one of the RRMs (RRM2). This allosteric phenomenon will be investigated using hydrogen-deuterium exchange mass spectrometry, fluorescence, and nonlinear optical spectroscopic methods. As the insert also offers a docking surface for regulatory chaperones, the effects of these proteins on SRPK1 function will be explored. The broader goal is to understand the SRPK1-ASF/SF2 assembly mechanism and to establish fundamental principles for splicing factor recognition and biological control within the larger SR protein family.
Since abnormalities in mRNA splicing have been linked to human disease, understanding the role of factors that actively participate in the splicing reaction may help us to develop treatments directed at the splicing machinery that can alleviate human suffering. SR proteins are critical factors whose phosphorylation controls where splicing takes place. Through analyses of the SR protein-enzyme complex, a better understanding of the link between SR proteins and disease may be attained.
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