Characterization of mRNA splicing factors : ASF/SF2 and Ire1p

Mechanisms of mRNA splicing greatly vary in complexity, ranging from simple reactions mediated by a two-component system to highly complex ones involving a macromolecular assembly called spliceosome. The former example of mRNA splicing is observed in the unfolded protein response (UPR) pathway, where intron excision is mediated by the kinase-endonuclease Ire1p and exon ligation is brought about by t-RNA ligase. Complex splicing reactions mediated by the spliceosome are more common and involve core ribonucleoprotein components and several accessory protein factors. The SR proteins are one such family of accessory factors. 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 can be extensively phosphorylated by SRPK and Clk/Sty kinases. The crystal structure of SRPK1 bound to an SR-protein derived peptide led to the identification of a docking motif on a prototype SR protein, ASF/SF2. The docking motif serves to restrict phosphorylation of ASF/ SF2 by SRPK1, thus generating a hypophosphorylated species which localizes to nuclear speckles. Subsequent hyperphosphorylation by Clk/Sty leads to its release from the speckles. We prove that docking motif is an important regulatory element for phosphorylation and subcellular localization of ASF/SF2. The X-ray crystal structure of the SRPK1:ASF/SF2 complex suggests that interactions between the kinase core of SRPK1 and the RRM2 domain of ASF/SF2 contribute to their high affinity interaction. We show that mutation of contact residues in ASF/SF2 affects its nuclear localization, but does not perturb its phosphorylation by SRPK1. We suggest that the regulation of ASF/SF2 by SRPK1 extends beyond that of a substrate by its kinase, and that SRPK1 acts as a chaperone for ASF/ SF2. We also investigated the RNA-binding property of ASF/ SF2 and found that, like other auxiliary splicing factors, ASF/SF2 binds specific sequence elements in mRNA with low affinity. We also show that in addition to both RRM domains, the inter-RRM linker contributes significantly to the affinity and specificity of RNA binding by ASF/SF2. Our interest in mRNA splicing and protein kinases led us to study Ire1p, which is a sensor and effector of UPR in yeast. Upon induction of UPR, Ire1p participates in a non- conventional splicing reaction to upregulate the transcription factor Hac1p, which induces UPR-responsive genes. We wanted to understand how the kinase and nuclease domains of Ire1p functionally interact with each other to orchestrate UPR, using X-ray crystallography as a tool. We have successfully purified and crystallized fragments of Ire1p, which encompass its kinase and nuclease domains. Attempts to determine the structure of the kinase-nuclease domain of Ire1p are currently underwa