|Budget Amount *help
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2003: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2002: ¥7,800,000 (Direct Cost: ¥7,800,000)
The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The selection of splice site can be altered by numerous extracellular stimuli, including growth factors, cytokines, hormones, depolarization, osmotic shock, and UVC irradiation, through synthesis, phosphorylation, and a change in localization of serine/arginine-rich (SR) proteins. SR proteins are a family of essential factors required for constitutive splicing of pre-mRNA and play an important role in modulating alternative splicing. They are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs (RRMs) at the amino terminus and an RS domain at the carboxy terminus.
Phosphorylation state of SR proteins appears to influence their activities in general and alternative splicing. To date, several kinases have been reported to phosphorylate SR proteins,
including SRPK-familykinases (SRPK1 and SRPK2), Clk/Sty family kinases, which consisted of four members (Clkl/Sty and Clk2-4), DNA topoisomerase I, p34cdc2 kinase and hPRP4, Addition of purified SRPK1 to permeabilized cells, or overexpression of SRPK1, 2 or one of CIk family members in transfected cells result in an apparent disassembly of the nuclear speckles.
The kinase inhibitor DRB (5,6-dichloro-1-β-D-ribo-furanosylbenzimidazole), which was previously shown to inhibit casein kinase II, inhibited Clk2 and induced the redistribution of SR proteins. Recently, through extensive screening of a chemical library, a specific inhibitor of Clkl/Sty and Clk4 was found and named as TG003. Clk 1/Sty-dependent splicing and disassembly of nuclear speckles were suppressed by TG003. Moreover, administration of TG003 rescued the embryonic defects induced by excessive Clk/Sty activity in Xen opus. As the importance of alternative splicing has been illustrated by the increasing number of human diseases attributed to missplicing events, TGOO3 will be applicable for the therapeutic manipulation of.abnormal splicing induced by activated Clk/Sty.
Considering the therapeutic application of inhibitors of SR protein kinases, prevention of virus is one of hopeful fields, because splicing is a crucial step for virus multiplication. In the case of HIV, 8 acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat and rev mRNAs, and the usage of these splicing sites are regulated by SR proteins and hnRNPs. In addition, Akusjarvi group reported that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. The anti-virus effects of inhibitors of SR protein kinases are now under investigation in our laboratory. Less