Proteome Analysis of Disease-related Genes based on Alternative Splicing

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(1) Computational analysis of disease-related genes with splice variants: We searched genes containing both ASDB (database of alternatively spliced genes) and OMIM (Online Mendelian Inheritance in Man) databases to extract alternatively spliced genes that related to diseases such as cancer and Alzheimer-disease.(2) Construction of a parallel IVV screening system: We optimized the conditions of IVV screening for protein-protein interactions (PPIs) in 96-well microplates with a QIAGEN BioRobot 8000. We also improved the performance of computer systems for analysis of the huge sequence data (from 1000 sequences per day to 16000 sequences per day).(3) A large-scale analysis of PPIs : As a result of the parallel IVV screening against 68 distinct bait proteins, total of -1000 PPIs were successfully detected. When arbitrary chosen PPIs were tested by pull-down assays, -70% of PPIs were verified.(4) Confirmation of PPIs with splice variants : Among the above PPI data, 24 gene fragments contain their protein-binding regions within variable regions derived from alternative splicing. We confirmed that the in vitro binding ability of these genes was certainly affected by the change of splicing patterns.(5) Construction of a PPI database : We developed a database containing not only the binding region from the PPI data but also the variable region of alternative splicing and SNP, the crystal structure of protein complexes, gene localization, etc. The database is useful for analyzing the effect of alternative splicing on disease-related gene networks.