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CERISE CERISE C. elegans C. elegans RNAi space RNAi space experiment” 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: [email protected] CERISE Mar. 26, 2009 @ JAXA

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Page 1: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

CERISECERISE““C. elegans C. elegans RNAi space experiment”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: [email protected] Mar. 26, 2009 @ JAXA

Page 2: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

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

Introduction of Introduction of Caenorhabditis elegansCaenorhabditis elegans

Page 3: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

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. Fire Craig C. MelloStanford Univ. Univ. of Massachusetts

Medical School

RNA interference: RNAi

nuceli

DNA

Cell

Ribosome: Translation

Protein

“ Targeting” specific gene silencing

mRNA degradation

Protein general

RNAi

Transcription: DNA>mRNA

Science overviewScience 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 However, it hasn’t been verified to work the RNAiRNAi in in space. space.

Page 4: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

Research objectivesResearch objectives

As objective , we propose to investigate whether Ⅰ RNAi occurs in space as the same manner as on the earth.

EarthEarth

Effects of space radiations

Effects of interaction of space radiations & g

11gg

Effects of g

11ggggSpaceSpace

Effects of g

CBEFCBEF

Credit: JAXA

Page 5: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

RNAi for OBJECTIVE ⅠRNAi for OBJECTIVE ⅠTwo 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

Page 6: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

Ce-RBX-1: an E3 ubiquitin ligase that is essential for meiosis, mitosis, and chromosomal condensation and segregation.

Ce-rbx-1 RNAi

Ce-rbx-1 RNAi

2 cells to 4 cells in WT

Page 7: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

OBJECTIVE ⅡOBJECTIVE ⅡEffects 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

Page 8: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

A: Before activation

B: Activation by removing U-pin in space

2 ml L1 larvae or dauers and 12 ml bacterial feeds

Culture systemCulture system

4 days: 1st generation

activation

8 days: 2nd-3rd generation

12 ml bacterial feeds

separated with U-pin

2 ml C. elegans

4- and 8-days culture at 20oC in space

1st generation: embryogenesis on earth2nd -3rd generation: embryogenesis in space

C: Termination by freezing in MELFI

Page 9: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

Culture bag Holder Meas Exp Unit A

16 bags 1 Holder

CBEF

4 Meas Exp Unit A(s)

Bio Dosimeter

Experimental flow for turn overExperimental flow for turn over

Page 10: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

Object Method

RNAi Cytological analyses

Real time RT-PCR

Gene and proteinexpression

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 analysisPost flight analysis

TsukubaTsukuba

JAXAJAXAAkira Higashibata Akira Higashibata et al. et al.

Page 11: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

PerspectivePerspective

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

Page 12: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

Credit: JAXA

Project Team MembersProject Team MembersTohoku UniversityJAXATohoku UniversityTohoku UniversityTohoku UniversityJAXAJAXAJAXAAESJSF

Atsushi HigashitaniAkira Higashibata

Mori ChihiroTakako Takanami

Nahoko HigashitaniTakashi YamazakiTomoko Sugimoto

Noriaki IshiokaToko Hashizume

Keiji Fukui

Page 13: CERISE “C. elegans RNAi space experiment” CERISE “C. elegans RNAi space experiment” RNA interference and protein phosphorylation in space environment using

AbbreviationAbbreviation

CBEF: cell biology experiment facilityPADLES: passive dosimeter for lifescience experiments in spaceRNAi: RNA interferenceRT-PCR: reverse transcriptase-polymerase chain reactionTLD: thermoluminescence dosimeter