| Budget Amount *help |
¥3,910,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Numerous genes in mammalian species undergo alternative polyadenylation, which results in transcripts with variable 3' end. Several lines of evidence suggest that poly(A) sites selection may be modulated developmentally or in a tissue specific manner, however, the fundamental mechanisms responsible for regulating alternative poly(A) site selection have not been elucidated. In this study, I showed involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3'- untranslated region (UTR). CFIm is a heterodimeric 3' end processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. I made polyclonal antibodies against GST fused 25 kDa subunit of CFIm (CFIm25) purified from E. coli extracts. CFIm25 was knocked down in HeLa cells by RNAi method and the knock down level was confirmed by western blotting by using the polyclonal antibody. To examine whether CFIm25 is involved in the regulation of poly(A) sites selection, I analyzed alternative poly(A) site selection of TIMP-2, syndecan 2, ERCC6 and DHFR genes by northern blotting. Results clearly showed changes in the distribution of mRNAs in CFIm25 depleted cells. Moreover, results in TIMP2, syndecan 2 and ERCC6 indicate that in the presence of CFIm the most upstream poly(A) sites in the terminal exons are not primarily used in HeLa cells, while in the absence of CFIm the most upstream poly(A) sites predominate. Therefore, this study suggests a role for CFIm in alternative poly(A) site selection. In the future, this study may support a novel gene therapy strategy by understanding mechanisms of poly(A) sites selection at a molecular level and contribute to basic science such as developmental biology and cell biology.
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