F.GENETIC STRAINS RESEARCH CENTER
F-g. Invertebrate Genetics Laboratory - Ryu Ueda Group

RESEARCH ACTIVITIES

(1) RNAi mutant fly bank for comprehensive analyses of gene function in Drosophila

Ryu Ueda, Misako Taniguchi, Yukiko Sado¹, Kaoru Saigo² and Kuniaki Takahashi (¹JST, ²Graduate School of Science, University of Tokyo)

--Genome sequencing projects have revealed the number of genes for several model organisms for genetics. The small worm Caenolabditis elegans, which is composed of only 959 cells, has 19,000 genes in its genome. On the other hand, Drosophila melanogaster, which has a long and sophisticated alimentary canal, a tubular heart that circulates hemolymph, and a large brain composed of over 10⁴ cells, harbors only 13,800 protein-coding genes. Considering there is such a small number of fly genes, each one of them may have an essential function in fly development and behavior. In other words, it may be easy to detect and analyze gene function in the fly by reverse genetics because the abnormal phenotype will frequently appear when knocking down a target gene whose function is unknown. We are planning to investigate the function of fly genes comprehensively as a suitable model for studying the functional genomics of multicellular organisms.
--How does one investigate the function of all 13,800 genes in the fly? We use RNA interference (RNAi) to knock down the activity of the target gene. RNAi is one of the emerging technologies with which to investigate gene function in multicellular organisms. When introduced into the cell, double stranded RNA (dsRNA) works as a specific mutagen for each gene. That is, dsRNA recognizes host mRNA and digests it in a sequence-specific manner, and consequently brings a loss-of-function mutation phenotype to the host cell. The detailed mechanism of this RNAi phenomenon has not yet been elucidated, but it works efficiently in many multicellular organisms, including humans.
--We coupled the RNAi with the GAL4-UAS gene expression system to induce a conditional loss-of-function mutation in the fly. The GAL4-UAS system is a binary system for inducing transgene expression, in which two fly lines are used. One is the GAL4 driver fly line, which expresses yeast transcription factor, GAL4, in a specific cell/tissue or at a specific developmental stage in favor of the GAL4 transgene. The other fly line harbors a transgene on the chromosome, in which an appropriate gene to be expressed is fused to the UAS promoter, the GAL4 target. When these two fly lines are crossed with each other, we can observe in the fly progeny that the GAL4 protein induces target transgene expression in a driver-specific conditional fashion. In this GAL4-UAS system, when we use a UAS-transgene having an inverted repeat (IR) sequence, the transcribed RNA may form a dsRNA in the cell and induce a loss-of-function mutation by the RNAi mechanism. Such inducible RNAi caused by the transcription of an IR sequence was first successfully adopted to gene function analysis in C. elegans. It was then also found to be effective in fly genetics. By making a UAS-transformation vector containing an IR sequence of the gene predicted by the fly genome project, and by introducing it into a fly line (IR fly), a mutant phenotype of the gene can be easily observed in any cell or at any developmental stage of the progeny, whenever the IR fly is crossed to an appropriate GAL4 driver fly.
--We are expanding this inducible RNAi to the whole genome of the fly. This process involves two major procedures.
1) in vitro construction of transformation vectors containing an IR sequence from each of the 13,800 predicted genes.
2) Transformation of IR vectors by injecting them into fly eggs and establishment of IR fly lines by traditional genetic methods.
--As of the end of 2004, over 6,900 transformation vectors had been constructed, 6,300 of which have been successfully introduced into the fly. We will continue this work in the next year, and may be able to add up more than 2,000 IR fly lines to our fly bank.
--Along with the establishment of IR fly lines, basic characterization of the target genes is conducted using these fly lines. All of the IR fly lines are crossed to the Act5C-GAL4 fly. The Act5C-GAL4 induces the UAS-transgene in all cells at all developmental stages. Thus, if the gene targeted by RNAi has functions that are indispensable for fly development, the progeny of IR and GAL4 flies should die before the adult flies emerge. Among the 1954 genes tested, 51.1% of the fly lines showed lethality. This value is rather high compared to that obtained by classical genetics (25%), while the fact that many of the genes tested here were considered to have important functions in various aspects of fly development by our collaborators may bring about such a high score. Detailed analyses on known genes and greater accumulation of data are necessary. We are currently collaborating with 70 groups. The usefulness of inducible RNAi for investigating gene function in Drosophila is being revealed in many aspects. We published 5 papers using RNAi flies^3)~7) and 2 papers on RNAi mechanism^{1), 2)} in 2004.
--This work was supported in part by financial assistance to Dr. Ueda from the Mitsubishi Kagaku Institute of Life Sciences (MITILS).

PUBLICATIONS

Papers
1. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R. and Saigo, K. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res., 32, 936-948.
2. Ui-Tei, K., Ueda, R., Zenno, S., Takahashi, F., Doi, N., Naito, Y., Yamamoto, M., Hashimoto, N., Takahashi, K., Hamada, T., Tokunaga, T. and Saigo, K. (2004). RNA interference induced by transient or stable expression of hairpin structures of double stranded RNA in Drosophila and mammalian cells. Molekulyarnaya biologiya, 38, 228-238.
3. Pili-Floury, S., Leulier, F., Takahashi, K., Saigo, K., Samain, E., Ueda, R. and Lemaitre, B. (2004). In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defence against Gram-positive bacterial infection in Drosophila adults. J. Biol. Chem. 279, 12848-12853.
4. Ichimiya, T., Manya, H., Ohmae, Y., Yoshida, H., Takahashi, K., Ueda, R., Endo, T. and Nishihara, S. (2004). The twisted-abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O-mannosyltransferase activity. J. Biol. Chem., 279, 42638-42647.
5. Kamimura, K., Rhodes, J.M., Ueda, R., McNeely, M., Shukla, D., Kimata, K., Spear, P.G., Shworak, N.W. and Nakato, H. (2004). Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase. J. Cell Biol., 166, 1069-1079.
6. Koganeya, A. K., Sasamura, T., Oshima, E., Suzuki, E., Nishihara, S., Ueda, R. and Hirabayashi, Y. (2004). Drosophila glucosylceramide synthase: A negative regulator of cell death mediated by proapoptotic factors. J. Biol. Chem., 279, 35995-36002.
7. Ishimaru, S., Ueda, R., Hinohara, Y., Ohtani, M. and Hanafusa, H. (2004). PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis. The EMBO J., 23, 3984-3994.

Reviews
8. Nishihara, S., Ueda, R., Goto, S., Toyoda, H., Ishida, H. and Nakamura, M. (2004). Approach for functional analysis of glycan using RNA interference. Glycoconj J., 21, 63-68.

ORAL PRESENTATIONS

1. Nishihara, S., Hino, M., Yoshida, H., Sasaki, N., Goto, S., Toyoda, H. and Ueda, R. Functional glycomics using Drosophila RNAi system. The US-Japan Glyco 2004, Hawaii, Dec. 2004.
2. 草間さきく,上田龍,須田健,西原祥子,松浦悦子「RNAi法によるショウジョウバエのSir2およびSir2-like遺伝子の機能解析」日本遺伝学会第76回大会,大阪,2004年9月.

POSTER PRESENTATIONS

1. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., Ueda, R. and Saigo, K. siRNA sequence requirement for effective RNA interference in mammalian cells and chick embryos. HGM2004, Berlin, Apr. 2004.
2. Ichimiya, T., Manya, H., Ohmae, Y., Yoshida, H., Ueda, R., Endo, T. and Nishihara, S. The functional analysis of Drosophila protein O-mannosyltransferases. The 77^th Annual Meeting of the Japanese Biochemical Society, Yokohama, Oct. 2004.
3. Ichimiya, T., Manya, H., Ohmae, Y., Yoshida, H., Ueda, R., Endo, T. and Nishihara, S. The functional analysis of Drosophila protein O-mannosyltransferases using RNAi mutant flies. The US-Japan Glyco 2004, Hawaii, Dec. 2004.
4. Michael Mende^G1, Tobias Bökers^G2, Hisataka Sabe^J1, Arul SubramanianI, Ryu Ueda^J2, Talila VolkI, Ryohei Yagi^J1, Andreas Prokop^G1.
5. 常泉和秀,菅野周平,上田龍,西郷薫,多羽田哲也,松本正吾「サイズ調節に関与するDrosophila nug1(dnug1), dnug2と相互作用する新規遺伝子の探索」日本発生生物学会第37回大会,名古屋,2004年6月.
6. 廣田ゆき,澤本和延,上田龍,岡野栄之「Transmembrane protein Tincar is expressed during and involved in the development of Drosophila photoreceptor cells」日本発生生物学会第37回大会,名古屋,2004年6月.
7. 細野千恵,松田亮,程久美子,上田龍,西郷薫「ショウジョウバエ複眼を用いた新規RNAi因子のスクリーニング」第27回日本分子生物学会年会,神戸,2004年12月.
8. 従二綾,程久美子,上田龍,程肇,西郷薫「マウスES細胞におけるDicerのノックダウン」第27回日本分子生物学会年会,神戸,2004年12月.
9. 程久美子,内藤雄樹,高橋史峰,原口健,従二綾,上田龍,西郷薫「RNAi効果の高いsiRNA配列設計ガイドライン」第27回日本分子生物学会年会,神戸,2004年12月.
10. 奥村美江子,井田寛之,吉田英樹,上田龍,坂口謙吾,山口政光「ショウジョウバエDNAポリメラーゼεの機能解析」第27回日本分子生物学会年会,神戸,2004年12月.
11. 大前佳子,吉田英樹,飯田真巳,豊田英尚,上田龍,西原祥子「ショウジョウバエグルクロン酸転移酵素群の機能解析」第27回日本分子生物学会年会,神戸,2004年12月.
12. 一宮智美,萬谷博,大前佳子,吉田英樹,上田龍,遠藤玉夫,西原祥子「ショウジョウバエO-マンノース転移酵素（POMT:protein O-mannosyltransferase）のRNAi knock down体を用いた機能解析」第27回日本分子生物学会年会,神戸,2004年12月.
13. 常泉和秀,菅野周平,上田龍,西郷薫,松本正吾「Drosophila nug1(dnug1), dnug2およびPi3kシグナル伝達経路と相互作用する新規遺伝子の探索」第27回日本分子生物学会年会,神戸,2004年12月.
14. 山本（日野）美紀,桑原玲子,原口朱夏,日下勇,服部成介,上田龍,西原祥子,後藤聡「糖鎖修飾の制御メカニズムを解明するゲノムワイドなスクリーニング系の構築」第27回日本分子生物学会年会,神戸,2004年12月.
15. 粟崎健,巽良子,高橋邦明,上田龍,伊藤啓「ショウジョウバエ変態期における神経-グリア相互作用による軸策分岐除去の機構」第27回日本分子生物学会年会,神戸,2004年12月.
16. 吉田英樹,上田龍,後藤聡,西原祥子「ショウジョウバエ神経発生におけるα 1-3フコース転移酵素の機能解析」第27回日本分子生物学会年会,神戸,2004年12月.
17. 高橋邦明,谷口美佐子,佐渡由希子,加藤直子,畑中宏美,粟野若枝,島村理恵子,森川由美子,小嶋徹也,善野修平,内藤雄樹,西郷薫,上田龍「ショウジョウバエにおける誘導型RNAi変異体バンク」第27回日本分子生物学会年会,神戸,2004年12月.

EDUCATION

1. R. Ueda was invited to give a seminar on “RNAi mutant fly bank" at Keio University, Tokyo, Mar., 2004 (in Japanese).
2. R. Ueda was invited to give a seminar on “RNAi technology" at the satellite meeting of the annual meeting of Japan society for Bioscience, Biotechnology, and Agrochemistry, Hiroshima, Mar., 2004 (in Japanese).
3. R. Ueda was appointed as an adjunct lecturer at Tsukuba University, Department of biological sciences to give a course on Genetics.

SOCIAL CONTRIBUTIONS AND OTHERS

1. R. Ueda joined advisory committee of National Institute of Agrobiological Sciences at Tsukuba.