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 is een ontvanger van een beurs &quot;Pierre Durand&quot; van de faculteit geneeskunde van de Universite Laval. Dit werk werd ondersteund door de Natural Sciences and Engineering Research Council of Canada (MOP-CG095386) naar RM De polysome fractionator werd verworven door een Canadese Stichting voor Innovatie subsidie ​​(MOP-GF091050) naar RMR M heeft een nieuw CIHR onderzoeker salaris award. We zijn dankbaar voor Drs. E. Khandjian, I. Gallouzi, S. Di-Marco en A. Cammas voor nuttig advies.

Het protocol volgt de richtlijnen goedgekeurd door Laval Ethical Review Board. 1. Voorbereiding van de celculturen en Brain Manipulatie <ol><li> Zoogdier-en Drosophila Cellen <ol><li> Grow HeLa cervicale kankercellen en Schneider Drosophila embryonale cellen, zoals aanbevolen door de American Type Culture Collection. Werken met cellen bij een lage doorgang. </li><li> Cellaag om 80% confluentie op de dag van het experiment bereiken. Voor het beste result...

<ol>
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De polysome profielanalyse op sucrosegradiënten maakt meting van translatie-initiatie door het analyseren van de dichtheid van polysomen geïsoleerd uit cellen of weefsels 9,11-14. Deze techniek is de beste (zo niet de unieke) benadering van de translatie-initiatie in vivo te meten. Het wordt gebruikt om de translationele positie van groeiende cellen tijdens de celcyclus 15 volgen en de effecten van verschillende soorten stress zoals virale infecties, hypoxie 13,16, 17 straling...

Eukaryote cellen voortdurend geconfronteerd met een aantal schadelijke fysiologische en ecologische stress omstandigheden die een snelle adaptieve cel respons vereisen. Cell spanningsreactie impliceert een nauwkeurige balans tussen anti-overleving en pro-survival werkende factoren. Het verstoren van dit evenwicht kan onomkeerbare gevolgen leiden van de ontwikkeling van de menselijke ziekten zoals kanker en neurodegeneratieve ziekten. Tijdens de eerste stap van de reactie op stress, cellen activeert pro-overleving routes die de gecoördineerde beheersing van veranderingen in genexpressie op het niveau van mRNA translatie omvatten. mRNA translatie bij eukaryoten is een complexe cellulaire proces dat gecoördineerde interactie tussen translatie initiatie factoren (EIFS), specifieke RNA-bindende eiwitten (SGD&#39;s), en RNA-moleculen 1 gaat. mRNA translatie is verdeeld in drie afzonderlijke fasen: initiatie, elongatie, en beëindiging. Hoewel alle drie de fasen zijn onderworpen aan Verordeninggevende mechanismen, translationeel controlemechanismen richten meestal de initiatieffase van de vertaling, die dus vormt de snelheidsbeperkende stap van eiwitsynthese 2. Translatie initiatie is een sterk geordende proces dat begint met de vorming van de eIF2a.GTP.Met-tRNA i Met ternaire complex en de daaropvolgende binding aan de 40S ribosoom subeenheid, leidt tot de vorming van het pre-initiatie complex. De volgende stap is de rekrutering van de preinitiation complex mRNA, die de activiteit van translatie initiatie factoren zoals eIF4F en eIF3 omvat. De aldus gevormde 48S preinitiation complex ondergaat specifieke conformationele verandering die dit mechanisme mogelijk te beginnen met het scannen van de 5&#39;-niet getranslateerde gebied van het mRNA totdat het startcodon AUG herkent. Het merendeel van de translatie initiatie factoren worden losgelaten en 60S subeenheden worden gerekruteerd voor een 80S vormen ribosoom complex bevoegd voor vertaling, eent welk punt eiwitsynthese gestart (figuur 1). Meer dan een 80S monosome kan vertalen hetzelfde mRNA tegelijk produceren van zogenaamde polysomen (of polyribosomen). De dichtheid van polysomen op mRNA weerspiegelt de initiatie, verlenging en beëindiging prijzen en is dus een maat voor de vertaalbaarheid van een bepaald transcript. Echter, polysoom profiel hoofdzakelijk gebruikt om veranderingen in mRNA translatie beoordeeld op de initiatie stap. Hier hebben wij een proteasoom inhibitor translatie initiatie remmer gebruikt. De behandeling van kankercellen met het medicijn veroorzaakt een stressreactie gekenmerkt door activering van de stress kinase genaamd HRI die de translatie initiatie factor eIF2a 3 fosforyleert. Fosforylering van eIF2a is een van de belangrijke gebeurtenissen die leiden tot de remming van translatie initiatie in zoogdiercellen 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

Nauwkeurige controle van mRNA translatie is fundamenteel voor eukaryote cel homeostase, met name in reactie op fysiologische en pathologische stress. Veranderingen van dit programma leiden tot de groei van beschadigde cellen, een kenmerk van kankerontwikkeling of voortijdige celdood zoals gezien in neurodegeneratieve aandoeningen. Veel van wat bekend is over de moleculaire basis voor translationele controlesignalen afkomstig is van polysome analyse met behulp van een dichtheidsgradiënt fractionering systeem. Deze techniek berust op ultracentrifugatie van cytoplasmatische extracten op een lineaire sucrose gradiënt. Wanneer de rotatie is voltooid, maakt het systeem fractionering en kwantificeren gecentrifugeerd zones die overeenkomen met verschillende vertalen ribosomen populaties, hetgeen resulteert in een polysome profiel. Veranderingen in de polysome profiel duiden op wijzigingen of defecten in translatie initiatie die optreden als gevolg van verschillende soorten stress. Deze techniek maakt het ook mogelijk om e te beoordelene rol van specifieke eiwitten op translatie-initiatie, en translationele activiteit van specifieke mRNA&#39;s te meten. Hier beschrijven we protocol polysome profielen voeren om translatie-initiatie van eukaryote cellen en weefsels te analyseren onder normale of spanning groeiomstandigheden.

Zoals vermeld polysoom profiel maakt de analyse van veranderingen van translatie initiatie onder stressomstandigheden. Figuur 1 is een vereenvoudigd aanzicht van translatie initiatie die als eerder een meerstaps proces waarbij een geordend samenstel van translatie initiatie complexen beschreven. Onder normale groeiomstandigheden, zijn translatie initiatie complexen omgezet in polyribosomen wiens opsporing door polysome profiel getuigen voor een actieve translatie initiatie (figuur 2; On...

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

Hier beschrijven we een werkwijze veranderingen in de initiatie van mRNA translatie van eukaryote cellen in reactie stresssituaties analyseren. Deze methode is gebaseerd op de snelheid scheiding op sucrosegradiënten vertalen ribosomen van niet-translating ribosomen.

Analyse van de translatie-initiatie tijdens stress Voorwaarden door polysome Profiling

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

Research

JoVE Journal

Biology

Monitoring Plant Hormones During Stress Responses

Monitoring plantenhormonen tijdens stress Reacties

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Antilichaam Profiling door Luciferase Immunoprecipitatie Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

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In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Analysis of Oxidative Stress in Zebrafish Embryos

Analyse van oxidatieve stress in zebravis embryo&#39;s

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profilering Individuele menselijke embryonale stamcellen door kwantitatieve RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

Polysome fractionering en analyse van zoogdieren Translatomes op een genoom-brede schaal

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Beoordeling van Selectieve mRNA Vertaling in zoogdiercellen door polysoom Profiling

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fecale Glucocorticoïd Analyse: Niet-invasieve bijnier Toezicht op Equids

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

Een eenvoudige methode voor Plant polysoomdisplays 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 van de Gap Junction-afhankelijke Transfer van miRNA met 3D-FRAP Microscopy

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