2006 Fiscal Year Final Research Report Summary
Construction of siRNA library for human functional genomics and hunting of RNAi-related genes.
| Project/Area Number |
16201040
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
基礎ゲノム科学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SAIGO Kaoru The University of Tokyo, Department of Biophysics and Biochemistry, Graduate School of Science, Professor, 大学院理学系研究科, 教授 (50136454)
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| Co-Investigator(Kenkyū-buntansha) |
UI-TEI Kumiko The University of Tokyo, Department of Biophysics and Biochemistry, Graduate School of Science, Associate Professor, 大学院理学系研究科, 助教授 (50213327)
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| Project Period (FY) |
2004 – 2006
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| Keywords | RNA interference / RNAi / siRNA / human / genome / functional genomics / siRNA library / RNAi-related gene |
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| Research Abstract |
RNA interference (RNAi) has been shown quite useful in the clarification of gene function in various organisms. Synthetic 21bp long double-stranded RNAs (dsRNAs) each with two 2nt long 3'overhangs have been found to serve as short interfering RNAs (siRNAs) for mammalian RNAi. The mechanisms of 21bp-long siRNA dependent RNAi in mammalian cells have been studied extensively, and we have established the rules for designing sequences of highly effective siRNAs. Our guidelines indicate 21bp long siRNAs simultaneously satisfying all four of the following sequence conditions to be capable of inducing highly effective gene-silencing in mammalian cells : A/U at the 5'end of the guide strand (GS) ; G/C at the 5'end of the passenger strand (PS) ; at least four A/U residues in the 5'terminal third of GS and the absence of any GC stretch of more than 9nt in length. RNAi is also known to be induced by the transfection of DNA encoding short hairpin RNA (shRNA). Large scale screening of loss-of-functi
… More
on mutants is possible if suitable shRNA-encoding DNA libraries are available. For construction of an shRNA-encoding DNA library, RNA-polymerase-III-promoter driven vectors have been widely used. But, this pol-III-driven system imposes various restrictions on shRNA sequences so that a considerable fraction of siRNA sequences becomes unavailable. To surmount this difficulty, we developed a new vector system which is driven by RNA polymerase II promoter. It is now possible to design any effective shRNA-encoding DNA without any sequence restrictions. I addition, we found that not only 21bp siRNA but also 22bp dsRNA is the final Dicer digestion product and showed some fraction of 22bp dsRNA to serve as an effective siRNA. Sequence preference rules for highly effective 22bp siRNA were very similar, if not identical, to those for 21bp siRNAs. We also found siRNA dimer and trimer to be capable of efficiently inducing RNAi when these oligomers possess two 2nt-long 3'overhangs and contain an active monomer unit in frame with respect to Dicer digestion. Thus, double- or triple-knockdown is possible in some suitable cell lines. Finally, we carried out gene screening experiments using our siRNA libraries and identified many candidates for human or mouse transcription-factor genes, apoptosis-related genes, and RNAi-related genes. Less
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Research Products
(45 results)
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[Journal Article] LARK activates posttranscriptional expression of an essential mammalian clock protein, PERIOD1.2007
Author(s)
Kojima, S., Matsumoto, K., Hirose, M., Shimada, M., Nagano, M., Shigeyoshi, Y., Hoshino, S., Ui-Tei, K., Saigo, K., Green, C.B., Sakaki, Y., Tei, H.
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Journal Title
Proc. Natl. Acad. Sci., USA. 104
Pages: 1859-1864
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells.2007
Author(s)
Sasaki, N., Sasamura, T., Ishikawa, H., Kanai, M., Ueda.R.Saigo, K., Matsuno, K.
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Journal Title
Genes Cells 12
Pages: 89-103
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Establishment of cell lines derived from the rat suprachiasmatic nucleus.2007
Author(s)
Kawaguchi, S., Shinozaki, A., Obinata, M., Saigo, K., Sakaki, T., Tei, H.
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Journal Title
Biochem. Biophys. Res. Commun. 355
Pages: 555-561
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Molecular cloning and characterization of a novel 3'-phosphoadenosin 5'-phosphosulfate transporter, PAPST2.2006
Author(s)
Kamiyama, S., Sasaki, N., Goda, E., Ui-Tei, K., Saigo K., Narimatsu, H., Jigami, Y., Kannagi, R., Irimura, T., Nishihara, S.
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Journal Title
J. Biol. Chem. 281
Pages: 10945-10953
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.2006
Author(s)
Kobayashi, M., Michaut, L., Ino, A., Honjo, K., Nakajima, T., Maruyama, Y., Mochizuki, H., Ando, M., Ghangrekar, I., Takahashi, K., Saigo, K., Ueda, R., Gehring, W.J., Furukubo-Tokunaga, K.
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Journal Title
Proc. Natl. Acad. Sci., USA 103
Pages: 14417-17722
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Aph-1 contributes to the stabilization and trafficking of the gamma-secretase complex through mechanisms involving inter-and intramolecular interactions.2005
Author(s)
Niimura, M., Isoo, N., Takasugi, N., Tsuruoka, M., Ui-Tei, K., Saigo, K., Morohashi, Y., Tomita, T., Iwatsubo, T.
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Journal Title
J. Biol. Chem. 280
Pages: 12967-12975
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] dsCheck : highly sensitive off-target search software for dsRNA-mediated RNA interference.2005
Author(s)
Naito, Y., Yamada, T., Matsumiya, T., Ui-Tei, K., Saigo, K., Morishita, S.
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Journal Title
Nucleic Acids Res. 33
Pages: W589-591
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila.2005
Author(s)
Takahashi, M., Takahashi, F., Ui-Tei, K., Kojima, T., Saigo, K.
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Journal Title
Development 132
Pages: 2547-2559
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Targeted expression of IP_3 sponge and IP_3 DSRNA impaires sugar taste sensation in Drosophila.2005
Author(s)
Usui-Aoki, K., Matsumoto, K., Koganezawa, M., Kohatsu, S., Isono, K., Matsubayashi, H., Yamamoto, M., Ueda, R., Takahashi, K., Saigo, K., Mikoshiba, K., Yamamoto, D.
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Journal Title
J. Neurogenetics 19
Pages: 123-141
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.2004
Author(s)
Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R., Saigo, K.
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Journal Title
Nucleic Acids Res. 32
Pages: 936-948
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] RNA-interference induced by transient or stable expression of hairpin structures of double-stranded RNA in Drosophila and mammalian cells.2004
Author(s)
Ui-Tei, K., Ueda, R., Zenno, S., Takahashi, F., Doi, N., Naito, Y., Yamamoto, M., Hashimoto, N., Takahashi, K., Hamada, T., Tokunaga, T., Saigo, K.
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Journal Title
Mol. Biol. 38
Pages: 276-287
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] siDirect : highly effective, target-specific siRNA design software for mammalian RNA interference.2004
Author(s)
Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S., Saigo, K.
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Journal Title
Nucleic Acids Res. 32
Pages: W124-129
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults.2004
Author(s)
Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K., Samain, E, Ueda, R., Lemaitre, B.
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Journal Title
J. Biol. Chem. 279
Pages: 12848-12853
Description
「研究成果報告書概要(欧文)」より
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