Name: analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro
Uploaded: 2014-05-03T15:00:00
Description: Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

SV er taknemmelig for finansieringen støtte fra NIH K22AI077353 og Landenberger Foundation. KR taknemmelig midler fra NIH (5SC1GM083754).

1.. Tilpasning af klæbende celler i Suspension (Dette er et valgfrit trin. Suspension celler generelt gøre bedre kerneekstrakter imidlertid celler dyrket i plader kan også anvendes.) <ol><li> Adapt adhærente celler til vækst i suspensionskultur under anvendelse af Joklik modificerede MEM suppleret med 5-10% nyfødt kalveserum eller føtalt bovint serum og 1% L-glutamin: penicillin-streptomycin. Propagate celler i rotationskolber med en filterhætte ved 37 ° C med 8% CO2. <...

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In vitro-præ-mRNA 3 &#39;kløvningsreaktionen udført i HeLa celle kerneekstrakter eller spaltningsprodukter faktorer fraktioneret fra disse ekstrakter, har gjort det muligt at identificere de centrale spaltningsprodukter faktorer og deres vigtigste komplekser 16-21. Mange flere proteiner forbundet med disse faktorer er for nylig blevet identificeret 22, og in vitro reaktionen kan fortsætte med at kaste lys over, hvordan disse proteiner bidrager til reaktionen. Måske fordi ...

Biosyntesen af ​​de fleste modne eukaryote besked RNA&#39;er (mRNA&#39;er), kræver flere posttranskriptionel modifikationer såsom capping, splejsning og polyadenylering. Disse ændringer er generelt forbundet for at sikre korrekt behandling 1 og kraftigt øge stabiliteten af mRNA. 3&#39;-enden dannelsen af pattedyr præ-mRNA&#39;er genereres ved spaltning i kernen af den spirende RNA efterfulgt af tilsætning af adenylat-rester i 5&#39;-spaltede produkt ved poly (A) polymerase (PAP) 2-4. I pattedyr er spaltning opnås ved en multikomponent-protein-kompleks, omtrent 800 kDa, som samler specifikke præ-RNA-sekvenser. Poly (A)-signalsekvens, et yderst konserveret kanonisk hexanukleotid sekvens AAUAAA dirigerer spaltningsstedet ved ca 10-30 nt nedstrøms. Dette site er specifikt anerkendt af spaltning og polyadenylering specificitet faktor (CPSF) og 73 kD underenheden afCSPF indeholder endonucleaseaktivitet. Spaltningen stimulering faktor (CstF) binder en mere degenereret GU-eller U-rige element sekvens nedstrøms for poly (A) site. Også påkrævet for spaltning pattedyr spaltning faktor I (CFIm) og pattedyr spaltning faktor II (CFII). CFIm binder de specifikke UGUA (N) steder i opstrømssekvensen elementer (bruger), der er defineret for en række gener, og synes at være involveret i vigtige fysiologiske processer 5-8. In vitro RNA-forarbejdning reaktioner almindeligvis analyseret ved anvendelse af radioaktivt mærkede RNA-substrater 9-12. Disse kan syntetiseres ved afstrømning transkription fra bakteriofag-promotoren T7 eller SP6. Når man studerer et polyadenyleringssted der er ikke blevet karakteriseret før, er det nødvendigt at anvende genomisk DNA snarere end cDNA til at generere RNA-substratet, som vigtige nedstrømssekvenser ikke kan være til stede i cDNA. Design substrater omfatte mindst 150 nt upstream og 50 nt nedstrøms fra spaltningsstedet / slut på den modne mRNA. Spaltningsproduktet migrerer hurtigere end substrat; men fordi andre fragmenter kan genereres ved ikke-specifik nuklease handling, specificitet af reaktionen skal verificeres af dets afhængighed af de korrekte behandling signalsekvenserne. Derfor RNA substrater med en punktmutation i AAUAAA-sekvens (f.eks AAGAAA) tjene som en negativ kontrol for spaltningsreaktionen. Da en lille mængde radioaktivt mærket RNA anvendes til spaltningsreaktionerne kan RNaser stede i høj overflod i de fleste kerneekstrakter være problematisk, og begrænse valget af udgangsmaterialet til ekstrakt forberedelse. HeLa-celler tendens til at indeholde lave niveauer af endogene RNaser og dermed klare sig godt i disse assays. Den endonukleolytisk spaltning af RNA-substrater in vivo og in vitro umiddelbart efterfølges af poly (A) tilsætning, thus den spaltede mellemprodukt er ikke til stede i påviselige mængder. Derfor, for at studere enten en specifik RNA-sekvens eller proteiner involveret i en spaltningsreaktion, er forsøg gennemføres under forhold, der forhindrer polyadenylering opstår. Der er ingen afhængighed af spaltning på polyadenylering, eller vice versa, så man kan stoppe polyadenylering uden at skade spaltningsreaktionen. Således er ATP erstattet med et kædeafsluttende analog, der mangler 3&#39;-hydroxylgruppe, således at kun et enkelt nucleotid kan inkorporeres i poly (A)-stedet og kun spaltet RNA kan påvises. I betragtning af kompleksiteten og høje grad af særpræg ved denne type assay, beskriver vi en detaljeret video-protokol til at studere endonukleolytisk spaltning ved spaltning / Poly (A) maskiner mRNA forstadier in vitro. Vi beskriver, hvordan man forbereder kompetente kerneekstrakter, generere radiomærkede RNA substrater, udføre kløvningsreaktionen, og analysere og fortolke resultatetdukter. Figur 1 viser et eksempel på substrat RNA&#39;er, der koder for 3&#39;-enden af HIV-1 pre-mRNA&#39;er, der skal anvendes til spaltning assay. 3&#39;-enden af HIV-RNA-genom er sammensat af mange vigtige regulatoriske sekvenser, såsom poly (A) site, et G+ U rig region, og USE element, som alle er nødvendige for en effektiv modning af virale mRNA-transkripter 13 . I dette eksempel ville vi forvente input RNA substrat til at være 338 nt og ved spaltning 237 nt. Hvis polyadenylering fik lov til at ske, vil der observeres en udstrygning af produkter mellem 237 og 437 nt.

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			<td height="21" style="height:21px;width:216px;">Expand High Fidelity PCR Kit</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">11 732 650 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mM dNTP Mix</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:119px;">Y02256</td>
			<td>10mM each nucleotide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MaxiScript SP6/T7 Kit</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM1322</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">m7G(5&#39;)ppp(5&#39;) G RNA Cap</td>
			<td style="width:141px;">New England Biolabs</td>
			<td style="width:119px;">S1404S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Century Marker Template Plus</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM7782</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easytides UTP [alpha-32p]-250uCi</td>
			<td style="width:141px;">Perkin Elmer</td>
			<td style="width:119px;">BLU507H250UC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading buffer II</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">8546G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC treated water</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">AM9906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x TAE</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP13354</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x TBE</td>
			<td style="width:141px;">Ameresco Life Sciences</td>
			<td style="width:119px;">0658-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP399-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP169-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Persulfate</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Acetate</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">A637-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40% 19:1 Acrylamide:Bis-acrylamide</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">161-0144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:141px;">Roche</td>
			<td style="width:119px;">10 901 393 001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100% Absolute Ethanol 200 Proof</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acid Phenol-Chloroform</td>
			<td style="width:141px;">Ambion</td>
			<td style="width:119px;">9720</td>
			<td>For RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintilation Fluid</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">SX18-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase Inhibitor</td>
			<td style="width:141px;">Promega</td>
			<td style="width:119px;">N261B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly (Vinyl alcohol) PVA</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Phosphate</td>
			<td style="width:141px;">Calbiochem</td>
			<td style="width:119px;">2380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100mM dATP</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP2560-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:141px;">Acros</td>
			<td style="width:119px;">23042-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">BP1700-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable Sequencing Unit</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351881-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Binder Clips</td>
			<td style="width:141px;">Office Depot</td>
			<td style="width:119px;">838-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx42cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352543</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20cmx22cm glass plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35252-7</td>
			<td>1SET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20cmx42cm Aluminum Cooling Plates</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352667</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx22cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35230-6</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4mmx42cm Spacers</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z352314-1</td>
			<td>1SET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z35195-4</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">16-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351962</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-well Comb</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z351970</td>
			<td>1EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Repel Coating</td>
			<td style="width:141px;">C.B.S. Scientific</td>
			<td style="width:119px;">SGR-0401</td>
			<td>or SGR-0101 for individual bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Loading Tips</td>
			<td style="width:141px;">Rainin</td>
			<td style="width:119px;">GT-10-4</td>
			<td>0.1-10uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sequencing PowerPac HV</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">PowerPac HV</td>
			<td>5000V/500mA/400W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Dryer Model 583</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;">Model 583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrotech Vacuum Pump for gel dryer</td>
			<td style="width:141px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glogos II Glow-in-the-dark Markers</td>
			<td style="width:141px;">Agilent</td>
			<td style="width:119px;">420201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 8x10</td>
			<td style="width:141px;">Midsci</td>
			<td style="width:119px;">BX810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film 14x17</td>
			<td style="width:141px;">Phenix</td>
			<td style="width:119px;">F-BX1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autoradiography Cassette</td>
			<td style="width:141px;">Fisher</td>
			<td style="width:119px;">FBCA 1417</td>
			<td>8x10 size available</td>
		</tr>
	</tbody>
</table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

3&#39;-enden af ​​mammale mRNA&#39;er dannes ikke ved brat ophør af transkription med RNA-polymerase II (RNPII). I stedet RNPII syntetiserer forløber mRNA efter udgangen af ​​modne RNA, og en aktiv proces endonucleaseaktivitet kræves på et bestemt websted. Spaltning af forstadiet RNA sker normalt 10-30 nt nedstrøms fra konsensus polyA stedet (AAUAAA) efter CA dinucleotider. Proteiner fra spaltning kompleks, en multifaktoriel protein kompleks af omtrent 800 kDa opnå dette specifikke nukleaseaktivitet. Specifikke RNA-sekvenser opstrøms og nedstrøms for poly-site kontrol rekrutteringen af ​​spaltningen kompleks. Umiddelbart efter spaltning, er præ-mRNAer polyadenylated af polyA polymerase (PAP) til at producere modne stabile RNA-beskeder. Behandling af 3&#39;-enden af ​​et RNA-transskript kan studeres ved hjælp af cellulære kerneekstrakter specifikke radiomærkede RNA-substrater. I summen, en lang 32 &lt;/ Sup&gt; P-mærket uspaltet precursor RNA inkuberet med kerneekstrakter in vitro, og spaltning vurderes ved gelelektroforese og autoradiografi. Når korrekt spaltning forekommer, en kortere 5&#39;spaltet detekteres produkt og kvantificeres. Her beskriver vi spaltningen assay i detaljer ved hjælp af, som et eksempel, 3&#39;-enden behandling af HIV-1 mRNA&#39;er.

Repræsentative resultater af en spaltning analyse af RNA-poly (A) områder af HIV-1 (figur 2). Vi kan se det uspaltede RNA-substrat, som er den langsomste vandrende bånd på toppen af ​​gelen. Den specifikke spaltede produkt er det mest intense kortere fragment band, der kører hurtigere i gelen ved den forventede størrelse, og er specielt fraværende fra spaltning analysen af ​​mutpoly (A) kontrol, som indeholder et punkt mutation i polyA sekvens (mutPolyA) RNA substrat. Nedbrydningsprodukte...

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA-polymerase II syntetiserer en precursor RNA, som strækker sig ud over 3&#39;-enden af ​​det modne mRNA. Den ende af det modne RNA genereres cotransskriptionelt på et sted dikteret af RNA-sekvenser via endonucleaseaktivitet af spaltning kompleks. Her har vi detaljeret metode til at studere spaltningsreaktioner in vitro.

Analyse af RNA Processing Reaktioner Brug Cell Free Systems: 3 &#39;End spaltning af præ-mRNA Underlag<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Research

JoVE Journal

Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Enkelt Læs og Forbundne End mRNA-Seq Illumina Biblioteker fra 10 nanogram Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transskription og lukning af Gaussia luciferase-mRNA Efterfulgt af HeLa Celletransfektion

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell specifik analyse af<em&gt; Arabidopsis</em&gt; Blade Med fluorescensaktiveret cellesortering

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrat Generation for endonukleaser af CRISPR / Cas Systems

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Metabolisk mærkning af Newly transskriberede RNA til High Resolution genekspressionsprofilering af RNA syntese, videreforarbejdning og Decay i Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

HeLa Baseret Cell ytringsfrihed Systemer til ekspression af<em&gt; Plasmodium</em&gt; Rhoptry Proteiner

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

En celle Gratis Assay til at studere Chromatin dekondensering i slutningen af ​​Mitose

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Påvisning af RNA-bindende proteiner ved<em&gt; In vitro</em&gt; RNA Pull-down i fedtcellerne Kultur

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...