Functional analysis of CF-I complex regulating mRNA 3' cleavage
| Project/Area Number |
18570159
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
WADA Tadashi Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, Associate Professor (60262309)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| 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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| Keywords | transcription / poly(A) site / mRNA / RNA cleavage / antibody / UTR / western blotting / northern blotting / 組換えタンパク質 / 免疫沈降 / RNA干渉 / 組織特異的 |
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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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Report
(3 results)
Research Products
(27 results)
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[Journal Article] Isolation of XAB2 complex involved in pre-mRNA splicing, transcripti on and transcription-coupled repair.2008
Author(s)
Kuraoka I, Ito S, Wada T, Hayashida M, Lee L, Saijo M, Nakatsu Y, Matsumoto M, Matsunaga T, Handa H, Qin J, Nakatani Y, Tanaka K.
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Journal Title
Journal of Biological Chemistry 283
Pages: 940-950
Description
「研究成果報告書概要(和文)」より
Related Report
Peer Reviewed
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[Journal Article] Isolation of XAB2 complex involved in premRNA splicing, transcription and transcription-coupled repair.2008
Author(s)
Kuraoka I, Ito S, Wada T, Hayashida M, Lee L, Saijo M, Nakatsu Y, Matsumoto M, Matsunaga T, Handa H, Qin J, Nakatani Y, Tanaka K
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Journal Title
J Biol Chem. 283
Pages: 940-950
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Drosophila Blimp-1 is a transient transcriptional repressor that controls timing of the ecdysone-induced developmental pathway.2007
Author(s)
Agawa Y, Sarhan M, Kageyama Y, Akagi K, Takai M, Hashiyama K, Wada T, Handa H, Iwamatsu A, Hirose S, Ueda H.
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Journal Title
Molecular and Cellular Biology 27
Pages: 8739-8747
NAID
Description
「研究成果報告書概要(和文)」より
Related Report
Peer Reviewed
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[Journal Article] Drosophila Blimp-1 is a transient transcriptional repressor that controls timing of the ecdysone-induced developmental pathway.2007
Author(s)
Agawa Y, Sarhan M, Kageyama Y, Akagi K, Takai M, Hashiyama K, Wada T, Handa H, Iwamatsu A, Hirose S, Ueda H
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Journal Title
Mol Cell Biol. 27
Pages: 8739-8747
NAID
Description
「研究成果報告書概要(欧文)」より
Related Report
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