Name: analysis of translation initiation during stress conditions by polysome profiling
Uploaded: 2014-05-19T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

PA er en mottaker av et stipend &quot;Pierre Durand&quot; fra Fakultet for medisin av Laval University. Dette arbeidet ble støttet av naturvitenskap og Engineering Research Council of Canada (MOP-CG095386) til RM The polysome fraksjone ble kjøpt gjennom en kanadisk Foundation for innovasjon stipend (MOP-GF091050) til RMR M har en ny CIHR etterforsker lønn award. Vi er takknemlige for legene. E. Khandjian, I. Gallouzi, S. Di-Marco og A. Cammas for nyttige råd.

Protokollen følger retningslinjene godkjent av Lavals Ethical Review Board. En. Utarbeidelse av cellekulturer og Brain Manipulasjon <ol><li> Pattedyr og Drosophila Cells <ol><li> Grow HeLa livmorhalskreftceller og Schneider Drosophila embryonale celler som anbefalt av American Type Culture Collection. Arbeid med celler på en lav passasje. </li><li> Plate-celler for å nå 80% sammenløp dagen for eksperimentet. For best resultat, bruk ~ 12 x 10 6...

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Den polysome profilanalyse på sakkarosegradienter tillater måling av oversettelse initiering ved å analysere tettheten av polysomes isolert fra celler eller vev 9,11-14. Denne teknikk er best (hvis ikke den unike) tilnærming til måling av translasjons-initierings in vivo. Den brukes til å overvåke statusen for translatorisk voksende celler i løpet av cellesyklusen 15, og for å vurdere virkningene av forskjellige typer stress, inkludert virusinfeksjoner, hypoksi 13,16, s...

Eukaryote celler stadig støter på en rekke skadelige fysiologiske og miljømessige stress forhold som krever rask adaptiv celle respons. Cell stressrespons innebærer en presis balanse mellom anti-overlevelse og pro-overlevelse virkende faktorer. Forstyrre denne balansen kan ha irreversible konsekvenser som fører til utvikling av menneskelige sykdommer som kreft og nevrodegenerative sykdommer. Under det første trinn av stressrespons, celler aktiverer pro-overlevelsesveier som involverer den koordinerte styring av endringer i genekspresjon på nivået av mRNA oversettelse. mRNA oversettelse i eukaryoter er en kompleks cellulær prosess som innebærer koordinert samhandling mellom oversettelse initiering faktorer (EIFS), spesifikt RNA bindende proteiner (RBDs), og RNA-molekyler en. mRNA oversettelse er delt inn i tre distinkte faser: initiering, forlengelse, og terminering. Selv om alle tre fasene er underlagt regulatory mekanismer, translasjonelle kontrollmekanismer retter seg for det meste initieringsfasen av translasjon, som således utgjør det hastighetsbegrensende trinn i proteinsyntesen 2. Oversettelse initiering er en høyt organisert prosess som begynner med dannelsen av eIF2a.GTP.Met-tRNA i Met ternært kompleks og etterfølgende binding til det 40S ribosom-underenheten, som fører til dannelsen av for tidlig detonasjon kompleks. Det neste trinnet er rekrutteringen av preinitiation komplekset til mRNA, noe som innebærer at aktiviteten av translasjon initiering av faktorer som eIF4F og eIF3. Den 48S preinitiation kompleks dermed dannet gjennomgår bestemte konformasjonsendringer som gjør dette maskineriet skal begynne å skanne 5&#39;-untranslated region av mRNA før den gjenkjenner startkodonet august De fleste av oversettelses initiering faktorene blir deretter løslatt og 60S subenheter er rekruttert for å danne en 80S ribosom kompleks kompetent for oversettelse, ent hvilket punkt proteinsyntese begynner (fig. 1). Mer enn en 80S monosome kan oversette det samme mRNA gangen produsere såkalte polysomes (eller polyribosomes). Tettheten av polysomes på en mRNA reflekterer initiering, tøyelighet og terminerings prisene og dermed er et mål på translatability av et bestemt transkript. Imidlertid er polysome profilen hovedsakelig brukt for å vurdere endringer i mRNA oversettelse ved initiering trinn. Her har vi brukt en proteasominhibitor som oversettelse initiering inhibitor. Behandling av kreftceller med dette stoffet induserer en stressrespons, karakterisert ved aktivering av stress kinase heter HRI som fosforylerer translasjons-initierings-faktor eIF2a 3. Fosforylering av eIF2a er en av de viktigste hendelser som fører til hemming av translasjon initiering i pattedyrceller 4.

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			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
			<td height="21" style="height:21px;width:279px;">Cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">FBS</td>
			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Sucrose solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">D-Sucrose</td>
			<td>Fisher Scientist</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">RNA Extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenol: Chloroforme</td>
			<td>Fisher Scientist</td>
			<td style="width:119px;">BP1754I-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
			<td style="width:119px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation initiation during stress conditions by polysome profiling

Nøyaktig kontroll av mRNA oversettelse er fundamental for eukaryot celle homeostase, særlig som følge av fysiologisk og patologisk stress. Endringer av dette programmet kan føre til vekst av skadede celler, et kjennetegn på kreftutvikling, eller til for tidlig celledød, for eksempel som vist i neurodegenerative sykdommer. Mye av det som er kjent angående den molekylære basis for translasjons-kontroll er blitt oppnådd fra polysome analyse ved hjelp av en densitetsgradient fraksjoneringssystem. Denne teknikken bygger på ultracentrifugation av cytoplasmatiske ekstrakter på en lineær sukrosegradient. Når spinningen er fullført, kan systemet fraksjonering og kvantifisering av sentrifugert soner svarende til forskjellige populasjoner sette ribosomer, noe som resulterer i en polysome profil. Endringer i polysome profilen er en indikasjon på endringer eller mangler ved translasjons-initierings som forekommer som respons på forskjellige typer stress. Denne teknikken gjør det også mulig å vurdere the rollen til spesifikke proteiner for translasjonsinitiering, og for å måle translatorisk aktiviteten av spesifikke mRNA. Her beskriver vi vår protokoll for å utføre polysome profiler for å vurdere oversettelse initiering av eukaryote celler og vev enten under normale eller stress vekstvilkår.

Polysome profilen Som nevnt ovenfor, tillater analyse av endringer av translasjons-initierings henhold stressbetingelser. Figur 1 er en forenklet visning av translasjon initiering som er beskrevet tidligere er en flertrinns prosess som omfatter en ordnet samling av translasjons-initierings-komplekser. Under normale vekstvilkår, er oversettelse initiering komplekser konvertert til polyribosomes som deteksjon av polysome profil attest for en aktiv oversettelse initiering (figur 2; Ubehan...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Her beskriver vi en fremgangsmåte for å analysere endringer i initiering av mRNA oversettelse av eukaryote celler som svar på stressbetingelser. Denne metoden er basert på hastighetsseparasjon på sakkarosegradienter av sette ribosomer fra ikke-sette ribosomer.

Analyse av Oversettelse Innvielse Under stressbetingelser ved Polysome Profilering

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Research

JoVE Journal

Biology

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Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

An Easy Method for Plant Polysome Profiling

En enkel metode for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Analyse av Gap Junction-avhengig Overføring av miRNA med 3D-FRAP Mikroskopi

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...