1. CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using the nematode Caenorhabditis elegans. PI: Atsushi HIGASHITANI Graduate School of Life Sciences, Tohoku University E-mail: ahigashi@ige.tohoku.ac.jp CERISE Mar. 26, 2009 @ JAXA

2. The advantages of working with C. elegans are (1) short life cycle (3.5 days at 20 o C), (2) entire genome sequenced in which about 35% of genes have human homologs, and (3) cell lineage identified completely. Introduction of Caenorhabditis elegans

3. The Nobel Prize in Physiology or Medicine 2006. "for their discovery of RNA interference - gene silencing by double-stranded RNA using the nematode C. elegans" Andrew Z. FireCraig C. Mello Stanford Univ.Univ. of Massachusetts Medical School Science overview RNAi technology performs in lots of life science researches, currently, has been applied several clinical examinations. However, it hasn’t been verified to work the RNAi in space.

4. Research objectives As objective Ⅰ, we propose to investigate whether RNAi occurs in space as the same manner as on the earth. Earth Effects of space radiations Effects of interaction of space radiations &  g 1g1g1g1g Effects of  g 1g1g1g1g  g Space Effects of  g CBEF Credit: JAXA

5. RNAi for OBJECTIVE Ⅰ Two target genes to monitor RNAi activity in space Recombinant GFP (Green fluorescent protein) gene Ce-RBX-1: an E3 ubiquitin ligase gene Reduction of green fluorescence by feeding dsRNA of GFP

6. Ce-RBX-1: an E3 ubiquitin ligase that is essential for meiosis, mitosis, and chromosomal condensation and segregation. Ce-rbx-1 RNAi 2 cells to 4 cells in WT

7. OBJECTIVE Ⅱ Effects of microgravity on entire gene and protein expressions, and protein phosphorylations beyond generations. Muscle atrophy and degradation Signal transduction against stresses under space environments Epigenetic modification

8. A: Before activation B: Activation by removing U-pin in space 2 ml L1 larvae or dauers and 12 ml bacterial feeds Culture system 4 days: 1 st generation activation 8 days: 2 nd -3 rd generation 12 ml bacterial feeds separated with U-pin 2 ml C. elegans 4- and 8-days culture at 20 o C in space 1 st generation: embryogenesis on earth 2 nd -3 rd generation: embryogenesis in space C: Termination by freezing in MELFI

9. Culture bagHolder Meas Exp Unit A 16 bags1 Holder CBEF 4 Meas Exp Unit A(s) Experimental flow for turn over

10. ObjectMethod RNAiCytological analyses Real time RT-PCR Gene and protein expression DNA micro array Proteome analyses Western blot analysis Epigenetic modification DNA methylation analysis Space radiations PADLES (Passive dosimeter for lifescience experiments in space) Post flight analysis Tsukuba JAXA Akira Higashibata et al.

11. Perspective Finally, the experimental results from flight samples might clarify the verification of RNAi activity and stress responses including muscle atrophy and alteration of gene and protein expressions under space environments. We expect that the data from this proposal will be useful for providing physiological and molecular biological effects of microgravity and clinical application of RNAi technology in space. Credit: JAXA

12. Project Team Members Tohoku University JAXA Tohoku University JAXA AES JSF Atsushi Higashitani Akira Higashibata Mori Chihiro Takako Takanami Nahoko Higashitani Takashi Yamazaki Tomoko Sugimoto Noriaki Ishioka Toko Hashizume Keiji Fukui

13. Abbreviation CBEF: cell biology experiment facility PADLES: passive dosimeter for lifescience experiments in space RNAi: RNA interference RT-PCR: reverse transcriptase-polymerase chain reaction TLD: thermoluminescence dosimeter