Recent studies have shown that a large family of splicing factors containing a serine/arginine-rich domain plays important roles in both constitutive and regulated splicing. The family members are generally referred to as SR proteins. SR proteins are phosphoproteins and increasing evidence suggests that they are targets for phosphorylation regulation in pre-mRNA splicing. To study this regulation, we have recently identified and cloned a cell cycle regulated kinase, SRPK1, that appears to be specific for SR proteins in mammalian cells. We have demonstrated that SRPK1 is responsible for the cell cycle dependent reorganization of a nuclear structure (the nuclear speckles) where SR proteins and other splicing factors are concentrated. Because a significant amount of SRPK1 is present in the nucleus in interphase cells, we focus in this proposal on investigating the interphase function and regulation of SRPK1.
Our specific Aim 1 is to address the function of SRPK1 in splicing by a series of in vitro experiments. We plan to determine the role of SRPK1 in splicing, and the effect of SR protein phosphorylation by SRPK1 on splice site selection. In addition, we will determine whether SRPK1 is a component of the spliceosome, and how SRPK1 is involved in splicing itself. Once the role of SRPK1 in splicing is established, we will, in Aim 2, address the regulation of SRPK1. Although SRPK1 contains a nuclear localization signal and a fraction of the kinase is present in the nucleus, a unique sequence in SRPK1 appears to function as a cytoplasmic retention signal that is responsible for the accumulation of a population of SRPK1 in the cytoplasm in interphase. Therefore, SRPK1 may be regulated by nuclear translocation. We plan to determine the minimal sequence required for the cytoplasmic localization of SRPK1 by deletion and linker scanning mutagenesis, and to identify a potential cytoplasmic anchor protein(s) that interacts with the retention signal. Further, we will examine the functional consequence of the nuclear translocation on splicing in vivo.
In Aim 3, we will study another mechanism of SRPK1 regulation by a specific inhibitor. We plan to purify the inhibitor and examine the mechanisms of inhibition. We will also investigate the role of the inhibitor in the regulation of splicing and splice site selection. Our ultimate goal is to explore the possibility that signals may be transduced through SRPK1,to regulate pre-mRNA splicing in the nucleus.
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