| Co-Investigator(Kenkyū-buntansha) |
KUROYANAGI Hidehito Tokyo Medical and Dental University, School of Biomedical Science, Lecturer, 大学院疾患生命科学研究部, 講師 (30323702)
FUKUHARA Takeshi Tokyo Medical and Dental University, Medical Research Institute, Research Associate, 難治疾患研究所, 助手 (20359673)
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| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2005: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2004: ¥8,500,000 (Direct Cost: ¥8,500,000)
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| Research Abstract |
Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes. This surprising finding underscores the importance of alternative splicing, through which a single gene can generate structurally and functionally distinct protein isoforms. Based on genome wide analysis, 75% of human genes are thought to encode at least two alternatively spliced isoforms. The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis.
A benzothiazole compound TG003, a kinase inhibitor that targets Clk1 and Clk4, suppressed dissociation of nuclear speckles, altered the splicing patterns, and rescued the embryonic defects induced by excessive Clk activity. The emerging inhibitors of the signal transduction pathways regulating pre-mRNA alternative sp
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licing may open the way to therapies against diseases caused by miss-splicing. We recently found that Clk is involved in the insulin-dependent alternative splising.
We identified a novel serine/arginine (SR)-rich-related protein as a binding partner of Clk4 mutant lacking kinase activity and designated it CLASP (Clk4-associating SR-related protein). CLASP is a binding partner of Clk4 and may be involved in the regulation of the activity of Clk kinase family. CLASP mRNA was highly expressed in brain, and both CLASP and Clk4 mRNAs were expressed in the hippocampus, the cerebellum, and the olfactory bulb. Two forms of CLASP were produced by a frameshift following alternative splicing. The staining of a short form of CLASP (CLASP-S) showed a nucleoplasmic pattern, while the long form of CLASP (CLASP-L) was localized as nuclear dots. Overexpression of CLASPL promoted exon EB inclusion from CR-1 and CR-2 pre-mRNA of Clk1. We are currently studying the role of CLASP on the survival of neuronal cells.
We have developed a transgenic alternative splicing reporter system that visualizes expression profiles of mutually exclusive alternative exons of a nematode C.elegans at a single cell level in vivo. We isolated mutant worms defective in the tissue-specific expression of the reporter and identified a novel evolutionarily conserved trans-acting factor, ASD-1 (alternative-splicing-defective-1), which also regulated tissue-specific alternative splicing of an endogenous gene. These results indicate that the reporter worm system can be used to analyze expression profiles of the alternative splicing isoforms, and to identify trans-acting factors and cis-acting elements. Furthermore, our results also demonstrated that regulation factors and elements of the tissue-specific alternative splicing events are conserved in vertebrates and nematodes.
Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. We originally developed SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. SRPIN340 suppressed propagation of HIV, Sindbis virus, SARS virus, and cytomegalovirus, suggesting that they may require SRPK-dependent SR protein phosphorylation for their multiplication. Less
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