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 takknemlig for økonomisk støtte fra NIH K22AI077353 og Landenberger Foundation. KR takknemlig erkjenner midler fra NIH (5SC1GM083754).

En. Tilpasning av heftende celler inn Suspension (Dette er et valgfritt trinn. Opphengs celler generelt ta bedre nukleære ekstrakter, men celler dyrket i plater kan også anvendes.) <ol><li> Tilpass adherente celler for vekst i suspensjonen ved hjelp av Joklik modifisert MEM supplert med 5-10% nyfødt kalveserum eller føtalt bovint serum og 1% L-glutamin: penicillin-streptomycin. Overfør cellene i spinnerflasker med et filterlokk ved 37 ° C med 8% CO2. </li><li> Ved tilpasni...

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Den in vitro-pre-mRNA 3 &#39;spaltningsreaksjon, utført i HeLa celle-ekstrakter eller atom med spalt-ningsfaktorer fraksjonert fra disse ekstrakter, har muliggjort identifisering av kjernen spalt-ningsfaktorer og deres hoved komplekser 16-21. Mange flere proteiner knyttet til disse faktorene har nylig blitt identifisert 22, og in vitro reaksjon kan fortsette å belyse hvordan disse proteinene bidrar til reaksjonen. Kanskje på grunn av reaksjonen in vivo synes å være c...

Biosyntesen av de fleste modne eukaryote melding RNA (mRNA) krever flere post-transcriptional modifikasjoner som tildekking, skjøting og polyadenylering. Disse modifikasjonene er generelt koplet for å sikre riktig behandling og en sterkt øke stabiliteten av mRNA. Den 3 &#39;ende dannelse av pattedyr-pre-mRNA er generert av endonucleolytic spaltning av den begynnende RNA, etterfulgt av tilsetningen av adenylat-rester til 5&#39; spaltet produkt av poly (A)-polymerase (PAP) 2-4. I pattedyr er spalting gjennomføres ved en multikomponent proteinkompleks, over ca 800 kDa, som setter sammen den spesifikke pre-RNA sekvenser. Det poly (A)-signal-sekvens, en høyt bevarte, anerkjente hexanucleotide sekvens AAUAAA, dirigerer spaltningssete ved omtrent 10 til 30 nt nedstrøms. Dette nettstedet er spesielt anerkjent av spalting og polyadenylering spesifisitet faktor (CPSF) og 73 kD subenhet avCSPF inneholder endonuclease aktivitet. Spaltingen stimuleringsfaktor (CstF) binder en mer degenerert GU-eller U-rik element sekvens nedstrøms for poly (A) område. Også som kreves for spalting er pattedyr spalting faktor I (CFIm) og pattedyr spalting faktor II (CFII). CFIm binder de spesifikke UGUA (N) nettsteder i oppstrøms sekvenselementer (bruker) som er definert for en rekke gener og synes å være involvert i viktige fysiologiske prosesser 5-8. In vitro, blir RNA-prosesseringsreaksjoner ofte analysert ved bruk av radioaktivt merkede RNA-substrater 9-12. Disse kan syntetiseres ved run-off transkripsjons fra bakteriofag T7 eller SP6 promoter. Når man studerer en polyadenylerings-sete som ikke er karakterisert ovenfor, er det nødvendig å bruke genom-DNA i stedet for cDNA for å generere den RNA-substrat, som viktige nedstrøms-sekvenser ikke kan være til stede i cDNA. Design underlag for å inkludere minst 150 nt ligger oppstrømsm og 50 nt nedstrøms fra spaltningssetet / slutten på den modne mRNA. Spaltingen produktet vandrer hurtigere enn substratet; Imidlertid, fordi andre fragmenter kan bli dannet ved ikke-spesifikk nuklease handling, har spesifisiteten av reaksjonen å bli bekreftet av sin avhengighet av de riktige prosesssignalsekvenser. Derfor RNA-substrater med en punktmutasjon i det AAUAAA sekvens (f.eks AAGAAA) tjener som en negativ kontroll for spaltning reaksjonen. Gitt at en liten mengde radiomerket RNA anvendes for spaltning reaksjoner, kan RNases stede i høy mengde i de fleste nukleære ekstrakter være problematisk, og begrenser valget av utgangsmaterialet for ekstrakt preparatet. HeLa-celler har en tendens til å inneholde lave nivåer av endogene RNases og således gi gode resultater i disse assays. Den endonucleolytic spalting av RNA-substrater in vivo og in vitro er umiddelbart etterfulgt av poly (A) addisjonssetet, tos den spaltet mellom er ikke til stede i målbare mengder. Derfor, for å studere enten en spesifikk RNA-sekvens, eller proteiner som er involvert i en kløyvingsreaksjon blir eksperimenter utført under forhold som hindrer polyadenylering oppstår. Det er ingen avhengighet av cleavage på polyadenylering, eller vice versa, slik at man kan stoppe polyadenylering uten å skade spaltningsreaksjon. Således er ATP erstattet med en kjede terminere analogt som mangler 3&#39;-hydroksyl-gruppen, slik at bare et enkelt nukleotid kan inkorporeres i poly (A) område og bare spaltes RNA kan detekteres. På grunn av kompleksiteten og høye grad av spesielle ved denne type assay, beskriver vi en detaljert video-protokoll for å studere endonucleolytic spalting ved spalting / poly (A) mRNA-maskiner av forløpere in vitro. Vi beskriver hvordan å forberede kompetente atom ekstrakter, generere radiomerket RNA underlag, utføre spaltningsreaksjon, og analysere og tolke resultateting produkter. Figur 1 viser et eksempel på substrat-RNA som koder for 3&#39;-enden av HIV-1 pre-mRNA for å brukes for en spalting assay. Den 3 &#39;ende av HIV-RNA genom er satt sammen av mange viktige regulatoriske sekvensene slik som poly (A) område, et G+ U rik region, og bruken element, som alle er nødvendige for effektiv modning av viral mRNA-transkripter 13 . I dette eksempelet vil vi forvente at innspill RNA underlaget å være 338 nt og ved spalting 237 nt. Hvis polyadenylering fikk lov til å forekomme, ville en smøre-produkter holdes mellom 237 og 437 nt.

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			<td style="width:119px;">D9938-1SET</td>
			<td>Different sizes available</td>
		</tr>
		<tr height="21">
			<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

Den 3 &#39;ende av pattedyr-mRNA&#39;er er ikke dannet ved brå avslutning av transkripsjon av RNA polymerase II (RNPII). I stedet syntetiserer RNPII forløper mRNA utover slutten av modne RNA, og en aktiv prosess med endonuklease-aktivitet er nødvendig ved en bestemt område. Spalting av forløperen RNA oppstår vanligvis 10-30 nt nedstrøms fra konsensus polyA side (AAUAAA) etter CA dinucleotides. Proteiner fra spalting komplekset, en multifaktoriell proteinkompleks på omtrent 800 kDa, oppnå dette spesifikke nuclease-aktivitet. Spesifikke RNA sekvenser oppstrøms og nedstrøms for polyA styre valg av rekrutteringen av spalting kompleks. Umiddelbart etter spalting, blir pre-mRNA polyadenylated av polyA-polymerase (PAP) til å produsere modne stabile RNA-meldinger. Behandling av 3 &#39;enden av et RNA transkript kan studeres ved hjelp av cellulære nukleære ekstrakter med spesifikke radioaktivt merkede RNA-substrater. I sum, en lang 32 &lt;/ Sup&gt; P-merkede uncleaved forløper RNA inkuberes med nukleære ekstrakter in vitro, og spalting bedømmes ved gelelektroforese og autoradiografi. Når korrekt spaltning inntreffer, en kortere 5 &#39;spaltes produktet blir detektert og kvantifisert. Her beskriver vi spalting assay i detalj ved hjelp av, som et eksempel, er 3 &#39;enden behandling av HIV-1 mRNA.

Representative resultater av et assay spalting av RNA-poly (A) områder av HIV-1 (figur 2). Vi kan observere uncleaved RNA-substrat, som er den langsomste migrere bånd på toppen av gelen. Den spesifikke spaltet produkt er den mest intense kortere fragment band som løper raskere i gelen med forventet størrelse, og er spesielt fraværende fra spalting analysen av mutpoly (A) kontroll som inneholder en punktmutasjon i polyA-sekvens (mutPolyA) RNA substrat. Nedbrytingsprodukter av inngangs underlaget ka...

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 forløper RNA som strekker seg utover den 3&#39;-enden av det modne mRNA. Den enden av det modne RNA genereres cotranscriptionally, på et sted diktert av RNA-sekvenser, via endonuklease aktivitet av spalting kompleks. Her har vi detaljer metoden for å studere cleavage reaksjoner in vitro.

Analyse av RNA prosessering reaksjoner Bruke Cell Free Systems: 3 &#39;End spalting av Pre-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 Les og Sammenkoblede Slutt mRNA-Seq Illumina Bibliotekene 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 Transkripsjon og tildekking av Gaussia luciferase mRNA Etterfulgt av Jakten Cell Transfeksjon

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 konkret analyse av<em&gt; Arabidopsis</em&gt; Leaves med fluorescens Aktivert Cell Sortering

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 av 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

Metabolsk Merking av ny-transkriberte RNA for High Resolution Gene Expression Profilering av RNA syntese, bearbeiding og Decay i cellekultur

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 Basert Cell ytringsfrihet Systemer for Expression of<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 Cell Gratis analysen å studere Chromatin Decondensation på slutten av Mitosen

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 av RNA-bindende proteiner etter<em&gt; In Vitro</em&gt; RNA Pull-down i adipocyte Culture

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 ...