Summary of Research Project Results under JSPS FY2002
"Research for the Future Program"

1.Research Institution Tokyo Medical and Dental University
2.Research Area Life Sciences
3.Research Field Regulation Networks of Eukaryotic Gene Expression
4.Term of Project FY 1998 - FY 2002
5.Project Number 98L01104
6.Title of Project Nuclear Structure and Gene Expression

7.Project Leader
Name Institution,Department Title of Position
Masatoshi, Hagiwara Tokyo Medical and Dental University, Medical Research Institute Professor

8.Core Members

Name Institution,Department Title of Position
Tsutomu, Ohta National Cancer Center, Genetics Division Section Head
Takashi, Ito Nagasaki University, School of Medicine Professor

9.Summary of Research Results

Transcription and Pre-mRNA splicing are closely coupled and catalyzed by a multimolecular complex including serine/argine-rich (SR) proteins, which are thought to play crucial role in the spliceosome formation and in the regulation of alternative splicing. SR proteins are RNA-binding proteins which have both one or two RNA-recognition motifs (RRMs) and serine/arginine repeats (RS domain), and are involved in constitutive splicing as well as being specific modulators in alternative splicing. In vivo, SR proteins are phosphorylated, predominantly on serine residues in the RS domain. Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian SRPKs and Clks, whose substrates are SR proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. We also found that a novel compound TG003, (Z)-1-(3-ethyl-5-methoxy-2, 3-dihydrobenzothiazol-2-ylidene) propan-2-one, inhibited AF2/ASF-dependent splicing in vitro by suppressing phosphorylation of SF2/ASF in Hela S100 extract. In vitro kinase assay revealed that TG003 specifically inhibited phosphorylation of SF2/ASF by Clk1/Sty and Clk4 with the Ki value of 10 nM. As for transcription factor CREB, we found that CaMKK-CaMKIV-CREB cascade is conserved in the sensory neurons of C. elegans, and that CRE::GFP expressing nematoda is a useful tool as an "in vivo reporter assay" system to visualize CREB activation in the living animal body.

10.Key Words
(1)mRNA factory   (2)SWI   (3)Chromatin
(4)Protein phosphorylation   (5)MR1   (6)p300