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 modtager af et stipendium &quot;Pierre Durand&quot; fra fakultetet for medicin Laval University. Dette arbejde blev støttet af naturvidenskab og teknik Forskningsråd Canada (MOP-CG095386) til RM polysom ​​fraktionator blev erhvervet gennem et canadisk Foundation for innovation tilskud (MOP-GF091050) til RMR M besidder en ny CIHR pris investigator løn. Vi er taknemmelige for Drs. E. Khandjian, I. Gallouzi, S. Di Marco og A. Cammas for nyttige råd.

Protokollen følger de retningslinjer, godkendt af Lavals Ethical Review Board. 1.. Udarbejdelse af cellekulturer og Brain Manipulation <ol><li> Pattedyr-og Drosophila-celler <ol><li> Grow HeLa cervical cancerceller og Schneider Drosophila embryonale celler som anbefalet af American Type Culture Collection. Arbejde med celler ved et lavt passage. </li><li> Plate-celler med henblik på at nå op på 80% konfluens på dagen for forsøget. For de bedste re...

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Den polysom ​​profil analyse på sucrosegradienter tillader måling af translationsinitiering ved at analysere tætheden af polysomer isoleret fra celler eller væv 9,11-14. Denne teknik er den bedste (hvis ikke den unikke) til at måle translationsinitiering in vivo. Det bruges til at overvåge translationel status voksende celler under cellecyklus 15, og for at vurdere effekten af forskellige typer af stress, herunder virusinfektioner, hypoxi 13,16, stråling 17,</sup...

Eukaryote celler konstant støder på en række skadelige fysiologiske og miljømæssige stress forhold, der kræver en hurtig adaptiv celle respons. Cell stress respons involverer en præcis balance mellem anti-overlevelse og pro-overlevelse handler faktorer. Forstyrre denne balance kan have uoprettelige konsekvenser, der fører til udvikling af humane patologier såsom cancer og neurodegenerative sygdomme. Under det første trin af stress respons, aktivere celler pro-overlevelse veje, der involverer koordineret kontrol af ændringer i genekspression på niveauet af mRNA translation. mRNA translation i eukaryoter er en kompleks cellulær proces, der involverer koordinerede samspil mellem translationsinitieringsregioner faktorer (EIFS), specifik RNA-bindende proteiner (RBDS) og RNA-molekyler 1. mRNA oversættelse er inddelt i tre særskilte faser: initiering, elongering og terminering. Selvom alle tre faser er underlagt reguleringlatory mekanismer translationelle kontrolmekanismer målrettet meste initieringsfasen oversættelse, som således udgør den hastighedsbegrænsende trin af proteinsyntese. 2. Oversættelse indvielse er en meget ordnet proces, der begynder med dannelsen af eIF2a.GTP.Met-tRNA jeg mødte ternære kompleks og den efterfølgende binding til 40S ribosom subunit, der fører til dannelsen af komplekset præ-indvielse. Det næste skridt er rekruttering af preinitiation kompleks til mRNA, som involverer aktiviteten af ​​translationsinitieringsregioner faktorer såsom eIF4F og eIF3. Den således dannede 48S preinitiation kompleks gennemgår specifikke konformationsændringer der muliggør dette maskineri til at begynde at scanne 5&#39;-utranslaterede region af mRNA, indtil den anerkender initieringskodonet august De fleste af de translationsinitieringsregioner faktorer er derefter frigivet og 60S-subunits er rekrutteret til at danne en 80S ribosomkomplekset kompetent til oversættelsen ent hvilket punkt Proteinsyntese starter (Figur 1). Mere end én 80S monosome kan oversætte den samme mRNA på et tidspunkt producerer såkaldte polysomer (eller polyribosomer). Tætheden af ​​polysomer på et mRNA afspejler den indvielse, strækning og termineringspriser, og dermed er et mål for oversættelighed af en bestemt udskrift. Imidlertid er polysom ​​profil hovedsageligt bruges til at vurdere ændringer i mRNA translation ved trin indledningen. Her har vi brugt en proteasominhibitor som translationsinitiering inhibitor. Behandling af cancerceller med dette stof inducerer en stress-respons er karakteriseret ved aktivering af stress kinase kaldet HRI som phosphorylerer translationsinitiering faktor eIF2a 3. Phosphorylering af eIF2a er en af de store begivenheder, der fører til hæmning af translationsinitiation 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

Præcis styring af mRNA-translation er grundlæggende for eukaryot celle-homeostase, især som reaktion på fysiologisk og patologisk stress. Ændringer i dette program kan føre til vækst i beskadigede celler, kendetegnende for udvikling af cancer, eller for tidlig celledød som ses i neurodegenerative sygdomme. Meget af hvad der er kendt om den molekylære basis for translationel er opnået kontrol fra polysom ​​analyse ved hjælp af en densitetsgradient fraktionering system. Denne teknik er baseret på ultracentrifugering af cytoplasmatiske ekstrakter på en lineær saccharosegradient. Når spin er afsluttet, giver systemet mulighed for fraktionering og kvantificering af centrifugerede zoner svarende til forskellige oversætte ribosomer befolkninger, hvilket resulterer i en polysom ​​profil. Ændringer i polysom ​​profil er tegn på ændringer eller mangler i translationsinitiering, der opstår som reaktion på forskellige former for stress. Denne teknik gør det også muligt at vurdere the betydningen af ​​specifikke proteiner på translationsinitiation og måle translationel aktivitet af specifikke mRNA&#39;er. Her beskriver vi vores protokol til at udføre polysom ​​profiler for at vurdere translationsinitiering af eukaryote celler og væv under enten normale eller stress vækstbetingelser.

Som tidligere nævnt, polysom ​​profil gør analysen af ændringer i translationsinitiering under stress betingelser Figur 1 er et forenklet billede af translationsinitiering der som tidligere beskrevet er en flertrins proces, der involverer en ordnet samling af translation initiation komplekser.. Under normale vækstbetingelser, er translationsinitieringsregioner komplekser omdannes til polyribosomer hvis påvisning ved polysom ​​nu vidner til en aktiv translationsinitiering (figur 2;</...

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 metode til at analysere ændringer i initiering af mRNA-translation af eukaryote celler som reaktion på stress-tilstande. Denne metode er baseret på hastigheden separation på saccharosegradienter med oversættelse ribosomer fra ikke-translating ribosomer.

Analyse af translationsinitieringskodon under stress betingelser ved Polysomprofiler Profiling

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

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