| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Alternative splicing (AltSp) of pre-mRNA is an important regulatory mechanism for quantitative control of gene expression and also for generating diverse protein species. The latter is now highlighted as the human genome sequencing project has revealed a limited number of genes (20,000-25,000) in the genome, which is far less than those expected for functional complexity. Studies on AltSp have focused on cassette-type (skipping) exons, however, we and others recently revealed widespread occurrence of another type of alternative splicing where the splice acceptor or donor site is selected between adjacent two. This type of AltSp makes a subtler difference, like 3 nucleotides in mRNA and a single amino acid residue in the protein product, thus we call it as "subtle alternative splicing". We have experimentally measured the ratio of the mRNA isoforms in more than 500 cases of Subtle AltSp. Our efforts have contributed to the connection between the bioinformatics and experimental fields. N
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ow, databases on the subject appear on internet, and several reports on respective genes suggest a functional significance of the isoforms.
It has been well known a considerable fraction of point mutations occurred in patients causes aberrant splicing. In some cases, a mutation near the canonical splice site abolishes the splice event using the site, and generates an aberrant splice product using a nearby cryptic site. With experiences with subtle AltSp, we assume that such aberrant splicing may be a consequence of competition between the nearby two sites, the canonical site modulated with the mutation and the cryptic site. Although many sites modulated with the mutation may loose competence as the site, other sites may retain some potential despite the mutation. We experimentally generated such sequences based on our previous results and tested the availability as the splice site. Several site sequences that were not used in the patient were indeed used when the cryptic site was further modified. This result suggests that the aberrant splicing in patients may be corrected once the cryptic splice site is modified. We have demonstrated that this indeed works by the use of RNA technology in experimental systems. Less
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