Former Professor, Dr. Masatoshi HAGIWARA, and his group members moved to Kyoto University in summer, 2010.

Dr. Hagiwara is now a professor of the Dept Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Here is a summary of researches that former Professor Hagiwara led in TMDU.

In the brain, many important molecules are regulated their structures and functions with this system, like signaling receptors, cell adhesion molecules, transcription factors, and so on. This mechanism contributes to the functional complexity and high performance of the mammalian brain through making divergent protein subsets from small number of genes. Also many neuronal diseases are caused by the mis-regulation of alternative splicing, i.e.; neurodegenerative disease, mental disorder, neuron-muscular dystrophy, or brain tumors. However, the mechanism of gene-, cell-, organ-, developmental stage-, and brain structure-specific regulation of alternative splicing is still mostly unknown.

We are developing a monitoring system of alternative splicing during the development of the brain using a fluorescent-based reporter vector system, and revealing their dynamism in single cell resolution (PLos ONE, 2010). With this system, we screen and select molecules that are essential for regulating alternative splicing, and exam their biological function during development (Fig. 2). This project will reveal the regulating mechanism of alternative splicing and its important roles from in vitro to in vivo.

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. SRPIN340 suppressed propagation of HIV, herpes simplex virus (HSV) type 1 and 2, Sindbis virus, SARS virus, and cytomegalovirus. These observations have led us to apply SRPIN340 to an antiviral drug. We are also going forward synthesis of series of SRPIN derivatives and testing the effect of these compounds on viral replication. Some SRPIN compounds dramatically inhibit the replication of hepatitis C virus, influenza virus, and Dengue virus.
Furthermore, we showed herpesvirus protein ICP27 changes the alternative splicing of promyelocytic leukemia protein (PML) pre-mRNA to affect virus replication (NAR, 2009). Our paper is first report showing the relationship between virus replication and host alternative splicing in detail.

We have previously reported the development of SRPIN340, a specific inhibitor of SR protein kinase (SRPK) family, and, TG003, a specific inhibitor of cdc2-like kinase (Clk) family. To date, they are the only specific inhibitors for SRPK and Clk, respectively. Significantly, we also proved that SRPIN340 is a potent anti-viral agent.

We are further developing novel inhibitors of other protein kinase families that are phylogenetically related to SRPK and Clk. SRPK family and Clk family are closely related protein kinase families, and they constitute a larger family of phylogenetically related protein kinases together with dual-specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family, Homeodomain interacting protein kinase (HIPK) family, and human pre-messenger RNA processing 4 protein kinase (PRP4). We propose to call the entire family as PSYCHIK family (PRP4, SRPK, DYRK, Clk, HIPK Family). These kinases are suggested to play important physiological roles including development and normal functioning of central nervous system, regulation of apoptosis, and pre-mRNA splicing.

We focus on PSYCHIK family members as targets for discovery of specific inhibitors, and indeed some novel specific inhibitors have been obtained.

We have characterized the new compounds through 1) in vitro assays, 2) in cell functional assays, 3) X-ray crystallography, and 4) whole embryo development assay. The findings demonstrated that the compounds are not only useful biological tools, but are potential drug seeds for hitherto untreatable diseases (Nat Commun, 2010).

Cells are often exposed to circumstance changes and various kinds of stresses such as canceration, viral infection, hypoxia condition, heat shock and radical toxicity. As reported by many researchers, various kinds of genes are regulated their expressions and functions by stress-responsive change of alternative splicing. However, the mechanisms underlying the stress-dependent splicing regulations are poorly understood. In order to reveal the molecular mechanisms, we utilize our splicing reporter system with model genes, whose splicing patterns are affected by several stress conditions.

Under stress condition, processes of gene expression such as transcription, pre-mRNA processing and translation are suppressed to avoid production of abnormal proteins. We found a novel mode of splicing promoted under stress conditions, when conventional splicing is generally arrested, and that a gene undergoes this stress-induced splicing serves to quick recovery of the cell after stress removal.