Name: isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution
Uploaded: 2017-01-16T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

Gli autori ringraziano il supporto dal NIH P50GM103297, P30CA100730 e Comprehensive Cancer P01CA16058.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Composizioni Buffer </td></tr><tr><td> Tampone di lavaggio: </td></tr><tr><td> 50 mM Tris-HCl, pH 7.4 </td></tr><tr><td> 150 mM NaCl </td></tr><tr><td> 3 mM MgCl2 </td></tr><tr><td> Basso sale Buffer: </td></tr><tr><td> 20 mM Tris-HCl, pH 7.5 </td></tr><tr><td> 10 mM NaCl </td></tr><tr><td> 3 mM MgCl2 </td></tr><tr><td> 2 mM DTT </td></tr><tr><td> 1x inibitore della proteasi cocktail senza ...

<ol>
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L&#39;isolamento RNP e la strategia di identificazione RNA cognate qui descritto è un mezzo selettivo di indagare una specifica interazione RNA-proteine ​​e di scoperta di proteine ​​candidate co-regolamentazione di un RNP specifica nelle cellule. Il vantaggio di utilizzare oligonucleotide-diretto RNase H scissione per isolare RNP è la capacità di catturare e analizzare il modo specifico cis-agendo elemento RNA RNP oltre RNP eterogenei legati a valle con l&#39;elemento RNA cis-age...

genica post-trascrizionale è regolato con precisione, cominciando con la trascrizione del DNA nel nucleo. Controllato da RNA vincolante proteine (RBPs), mRNA biogenesi e il metabolismo si verificano in particelle ribonucleoproteiche altamente dinamici (RNP), che associano e dissociano con un mRNA substrato precursore durante la progressione di RNA metabolismo 1-3. cambiamenti dinamici nella componenti RNP influenzano il destino post-trascrizionale di mRNA e di fornire la garanzia della qualità durante la lavorazione dei trascritti primari, i loro traffici nucleari e la localizzazione, la loro attività come modelli di mRNA per la traduzione, e l&#39;eventuale fatturato di mRNA maturi. Numerose proteine sono designati come RBPs in virtù dei loro domini ammino acido conservato, tra cui il motivo di riconoscimento RNA (RRM), l&#39;RNA a doppio filamento dominio di legame (REA), e tratti di residui basici (ad esempio, arginina, lisina e glicina) 4. RBPs sono di routineisolato da strategie di immunoprecipitazione e sono proiettati per identificare i loro RNA cognate. Alcuni RBPs co-regolare pre-mRNA che sono funzionalmente correlati, designati come regulons RNA 5-8. Questi RBPs, i loro mRNA cognate, e talvolta non codificante RNA, formano RNP catalitici che variano in composizione; loro unicità è dovuta a varie combinazioni di fattori associati, nonché al temporale sequenza, posizione, e la durata delle loro interazioni 9. RNA immunoprecipitazione (RIP) è una tecnica potente per isolare RNP dalle cellule e per identificare le trascrizioni associati con analisi di sequenza 10-13. Passando da candidato a Genome-wide screening è possibile attraverso RIP combinato con un&#39;analisi microarray 14 o high-throughput sequencing (RNA-Seq) 15. Analogamente, proteine co-precipitazione possono essere identificate mediante spettrometria di massa, se sono sufficientemente abbondanti e separabili dal anticorpi co-precipitazione 16,17. Qui, ci rivolgiamo la metodologia per isolare i componenti RNP di una specifica RNA affine da cellule umane in coltura, anche se l&#39;approccio è alterabile per lisati solubili di cellule vegetali, funghi, virus e batteri. Analisi a valle del materiale comprendono l&#39;identificazione candidato e validazione mediante immunoblot, spettrometria di massa, saggio enzimatico biochimico, RT-qPCR, microarray, e RNA-Seq, come riassunto in Figura 1. Dato il ruolo fondamentale della RNP nel controllare l&#39;espressione genica a livello post-trascrizionale, alterazioni nell&#39;espressione dei RBPs componenti o la loro accessibilità alle cognate RNA possono essere dannosi per la cellula e sono associati a diversi tipi di disturbi, malattie neurologiche 18. DHX9 / RNA elicasi A (RHA) è necessario per la traduzione di mRNA selezionati di origini cellulari e retrovirali 6. Questi RNA affini presentano strutturalmente affini elementi cis-agenti nel loro5 &#39;UTR, che è designato come elemento di controllo post-trascrizionale (PCE) 19. Attività RHA-PCE è necessaria per la traduzione efficiente cap-dipendente di molti retrovirus, compreso l&#39;HIV-1, e di geni regolatori crescita, tra Jund 6,20,21. Codificata da un gene essenziale (dhx9), RHA è essenziale per la proliferazione cellulare e la sua down-regolazione elimina la vitalità delle cellule 22. L&#39;analisi molecolare di RHA-PCE RNP è un passo essenziale per comprendere il motivo per cui l&#39;attività RHA-PCE è necessario controllare la proliferazione cellulare. La caratterizzazione precisa dei componenti RNP RHA-PCE allo stato stazionario o su perturbazione fisiologica della cella richiede l&#39;arricchimento selettivo e la cattura delle RNPs RHA-PCE in abbondanza sufficiente per l&#39;analisi a valle. Qui, retrovirale PCEgag RNA è stato etichettato con 6 copie del cis-acting sito di legame RNA per la proteina di rivestimento MS2 (CP) all&#39;interno della cornice di lettura aperta. La proteina di rivestimento MS2 è stato exogenously co-espresso con PCEgag RNA dal plasmide trasfezione per facilitare l&#39;assemblaggio RNP in cellule in crescita. RNP contenenti la proteina di rivestimento MS2 con cognate MS2-tag PCEgag RNA sono stati immunoprecipitati dall&#39;estratto cellulare e catturati su sfere magnetiche (Figura 2a). Per catturare selettivamente i componenti RNP legati alla PCE, l&#39;RNP immobilizzato è stato incubato con un oligonucleotide complementare alle sequenze distali alla PCE, formando un ibrido RNA-DNA che è il substrato per l&#39;attività RNasi H. Poiché PCE è posizionato in &#39;terminale 5&#39; del 5 untranslated regione, l&#39;oligonucleotide era complementare alle sequenze di RNA adiacenti al sito di inizio traduzione retrovirale (gag codone di inizio). RNase H scissione in prossimità della partenza gag codone rilasciato complesso UTR 5 &#39;dalla RNP immobilizzata, che è stato raccolto come eluente. Successivamente, il campione è stato valutato mediante RT-PCR per confermare la cattura di PCEgag e SDS PAGE e immunoblotting per confermare la capture della proteina di rivestimento bersaglio MS2. Una convalida del legame RNA proteina PCE-associata, DHX9 / RNA elicasi A, è stata quindi eseguita.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F7425</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RHA antibody</td>
			<td>Vaxron</td>
			<td>PA-001</td>
			<td></td>
		</tr>
		<tr>
			<td>TRizol LS reagent</td>
			<td>Life technology</td>
			<td>10296-028</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>Ambion</td>
			<td>AM2292</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26184</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaeasy clean-up column</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript reverse transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Out</td>
			<td>Invitrogen</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>98379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>17904</td>
			<td></td>
		</tr>
		<tr>
			<td>Random hexamer primers</td>
			<td>Invitrogen</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo-dT primers</td>
			<td>Invitrogen</td>
			<td>AM5730G</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>IDT</td>
			<td></td>
			<td>Gene specific primers for&#160; PCR amplification</td>
		</tr>
		<tr>
			<td>Oligonucleotide for RNase H mediated cleavage</td>
			<td>IDT</td>
			<td></td>
			<td>Anti-sense primer for target RNA</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco Life technology</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM tissue culture medium</td>
			<td>Gibco Life technology</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life technology</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S642-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-Rad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td></td>
			<td></td>
			<td>1.7 ml micro-centrifuge tube holding</td>
		</tr>
		<tr>
			<td>Laminar hood</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>Protein gel apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Protein transfer apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample transfer</td>
		</tr>
		<tr>
			<td>Ready to use protein gels (4-15%)</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td></td>
			<td></td>
			<td>Pellet down the sample</td>
		</tr>
		<tr>
			<td>Table top rotator</td>
			<td></td>
			<td></td>
			<td>Mix the sample end to end</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Mix the samples</td>
		</tr>
	</tbody>
</table>

isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

Risultati RIP precedenti identificati RNA gag retrovirali e selezionati RNA cellulari che la co-precipitato con DHX9 / RHA, compreso l&#39;HIV-1 6, Jund 6 e Hur (Fritz e Boris-Lawrie, inedito). Il retrovirale 5 &#39;UTR è stata dimostrata a co-precipitato con DHX9 / RHA nel nucleo e di co-isolare nel citoplasma in poliribosomi. Si è univocamente definita come la CSI -acting elemento di controllo post-trascrizionale (PCE) 6. Per isolare PCEgag R...

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Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Isolamento di cognate Complessi RNA-proteina da Cellule con eluizione oligonucleotidi-diretto

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Research

JoVE Journal

Biochemistry

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Caratterizzazione di complessi multi-subunità proteiche di MxA umano per mezzo di non-denaturare poliacrilammide gel elettroforesi

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Ricerca

Biochimica

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Tandem Affinity Purification di complessi proteici da cellule eucariotiche

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Rapid Isolation of the Mitoribosome from HEK Cells

Rapido isolamento dei Mitoribosome da cellule HEK

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

L'analisi di complessi proteici dinamico montato su e rilasciato da Biolayer interferometria biosensore utilizzando la microscopia elettronica e spettrometria di massa

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

Una procedura di isolamento del RNA basati su Oligonucleotide Tandem recuperare mRNA-proteina eucariotica complessi

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Purificazione di affinità del cloroplasto Translocon proteina complessi usando il Tag di rubinetto

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Isolation of Tissue Extracellular Vesicles from the Liver

Isolamento dei Tessuti Vescicoli Extracellulari dal Fegato

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

Traduzione della purificazione dell'affinità del ribosoma (TRAP) per l'isolamento dell'RNA dalle cellule endoteliali In vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

Pinzette ottiche per studiare le interazioni RNA-proteina nella regolazione della traduzione

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Estrazione di microtubuli modificati da cellule di mammifero per studiare complessi microtubulo-proteine mediante microscopia crioelettronica

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...