Name: desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells
Uploaded: 2018-04-25T15:00:00
Description: Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Dette arbejde blev støttet af den schweiziske National Science Foundation Sinergia grant CRSII3 147697, Stiftung für Angewandte Krebsforschung og Zürich Krebsliga.

1. forberedelse af cytosole og nukleare proteinfraktion
<ol><li>Plade 4 x 106 ACC-MESO-4 celler i en T150 celle kultur kolbe dyrkes i RPMI - 1640 medium suppleret med 10% FBS, 1 x penicillin/streptomycin (100 x) og 2 mM L-glutamin (200 mM). Når celler når et sammenløb af 80-90% (~5-5.5 x 106 celler), fortsætte med protein udvinding af nukleare og cytosole brøkdel. Bemærk: ACC-MESO-4 cellelinie blev indhentet fra RIKEN BioResource Center11.</li>
	<li>Opsug medium, v...

<ol>
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L., Yong, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+and+post-transcriptional+gene+regulation.">RNA-binding proteins and post-transcriptional gene regulation.</a> FEBS Letters. 582 (14), 1977-1986 (2008).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+biological+roles+of+alternative+3'UTRs.">Evolution and biological roles of alternative 3'UTRs.</a> Trends in Cell Biology. 26 (3), 227-237 (2016).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3'+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biology. 9 (5), 563-576 (2012).</li><li>von Roretz, C., Di Marco, S., Mazroui, R., Gallouzi, I. 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Magnetic bead technology for better assay development Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf">https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf</a> (2013)</li><li>Usami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+four+malignant+pleural+mesothelioma+cell+lines+from+Japanese+patients.">Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients.</a> Cancer Science. 97 (5), 387-394 (2006).</li><li>Laemmli, U. 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3' UTRs tilhører ikke-kodende genom3, og alle ikke-kodende RNA'er kan interagere med proteiner for at udøve deres funktion7. Når det mammale genom blev anset for at være korrekt transskriberet og produceret en betydelig del af lange noncoding RNA'er16, viste nye beviser, at disse lange-noncoding RNA'er fungere i reguleringen genekspression som de interagerer med kromatin-remodeling komplekser17. Denne viden blev opnået ve...

Genekspression og det endelige niveau for gen produktet kan reguleres tæt ved at påvirke mRNA stabilitet og mRNA oversættelse sats1. Disse post-transcriptional reguleringsmekanismer er udøvet gennem vekselvirkninger mellem ikke-kodende RNA og/eller RNA-bindende proteiner (RBPs) med målrettede mRNA. Det er normalt den 3' utranslaterede region af mRNA (3' UTR - tilhører den ikke-kodende del af genom2) der indeholder specifikke cis-regulerende elementer (CRE), som er anerkendt af trans-handler faktorer såsom miRNA eller RBPs 3. den bedst undersøgte cis-element inden for 3' UTR, er motivet er adenin-rige element (er), som er anerkendt af specifikke AU-bindende proteiner (AUBP), og til gengæld, inducerer enten mRNA nedbrydning/deadenylation (er-medieret decay) eller mRNA stabilisering4.
Størrelsen af 3' UTR af calretinin mRNA (CALB2) er 573 bp lang og indeholder en formodede AUUUA pentamer, som forudsagt af AREsite2, en bioinformatic værktøj5. Overensstemmelse med tilstedeværelsen af en formodet er motiv, pmirGLO vektor-reporter Analysen viste en stabilisering rolle af dette element inden for CALB2 mRNA6. Endelig, in vitro- RNA-pulldown blev brugt til at identificere den AUBP, der stabiliserer calretinin mRNA gennem er motiv.
Da alle ikke-kodende RNA'er interagerer med proteiner7, in vitro- RNA-pulldown er en god måde og første of choice analyse for at identificere RNA-interactors til støtte i afkodningen molekylære mekanismer. I denne RNA-pulldown metode, blev kommercielt syntetiserede RNA-sonder, der var mærket med en modificeret form af biotin (desthiobiotin) fra 3' endepunktet af RNA-streng, anvendt. RNA-desthiobiotin er immobiliseret gennem interaktion med streptavidin på magnetiske perler, som bruges til at trække ned proteiner, der specifikt interagerer med bundet-RNA af interesse. Ikke-denatureret og aktive proteiner fra det cytosole brøkdel af lungehindekræft celler der bruges som kilde til proteiner. Disse RNA-bundet proteiner er elueret fra de magnetiske perler, køre gennem en 12% SDS-PAGE gel, overføres til en membran og aftestede med forskellige antistoffer.
I standard streptavidin-biotin affinitet rensning procedure, barske denaturering betingelser er nødvendige for at forstyrre den stærke irreversibel biotin-streptavidin bånd for at eluere bundne proteiner8, hvilket kan føre til dissociation af proteinkomplekser. I modsætning til biotin, desthiobiotin reversibelt bindes til streptavidin og er konkurrencedygtig forskydes med en bufferopløsning af biotin, giver mulighed for en blid eluering af proteiner, og at undgå isolation af naturligt biotinylated molekyler9, tyder på, at teknikken er ideel til at isolere nativt proteinkomplekser indfødte betingelser.
In vitro bindende betingelser og strenghed, som bestemmes af saltkoncentration, reduktionsmiddel og vaskemiddel procentdel, bør være tæt på de tilstedeværende i forbindelse med cellulære for at identificere sande i vivo interaktioner. De bindende betingelser gennemført heri har tidligere påvist som passende for en identifikation af HuR som en AU-bindende protein10. Denne tilgang kunne spare tid, da optimering af ordentlig bindende betingelser kan være tidskrævende og udfordrende. Desuden, denne metode kunne anvendes som en start protokol for enhver RNA-pulldown eksperiment og gradvist kan optimeres ved at ændre koncentration af salte og rengøringsmidler, ændre glycerol procentdelen og tilføje andre salte. Vi viste desuden, at selv en kort 25-nukleotid RNA-sonde husly en pentamer er-motiv kan bruges til at vise interaktion med en specifik AUBP.

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

In vitro- RNA-pulldown anvendes stadig i vid udstrækning i de første trin af protokoller med henblik på at identificere RNA-bindende proteiner, der anerkender bestemt RNA strukturer og motiver. I protokollens RNA-pulldown kommercielt syntetiserede RNA-sonder er mærket med en modificeret form af biotin, desthiobiotin, fra 3' endepunktet af RNA-streng, der kan dekrypteres binder sig til streptavidin og dermed giver eluering af proteiner under mere fysiologisk betingelser. RNA-desthiobiotin er immobiliseret gennem interaktion med streptavidin på magnetiske perler, som bruges til at trække ned proteiner, der specifikt interagerer med RNA af interesse. Ikke-denatureret og aktive proteiner fra det cytosole brøkdel af lungehindekræft celler der bruges som kilde til proteiner. Metoden beskrives her kan anvendes til påvisning af samspillet mellem kendte RNA-bindende proteiner og en 25-nukleotid (nt) lang RNA sonde der indeholder en sekvens af interesse. Dette er nyttigt til at fuldføre den funktionelle karakterisering af stabiliserende eller destabiliserende elementer findes i RNA molekyler opnås ved hjælp af en reporter vektor assay.

I dette eksperiment, blev en 25-nt lang fragment af calretinin 3' UTR husly er motiv (CALB2 3' UTR (er) 25-nt) brugt til at teste, om det binder sig specifikt til den menneskelige-antigen Rasmussen (HuR) protein, en kendt mRNA stabilisator. For at teste specificiteten af elementet er, en 25-nt RNA var sonden CALB2 3' UTR (mtARE) indeholdende en er-motiv mutation, som tidligere blev vist at afskaffe stabilisering effekten er motiv, brugte6. Den tredje RNA sonde repr...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-Streptavidin-affinitet medieret oprensning af RNA-interagere proteiner i Mesotheliom celler

Desthiobiotin mærkning af en syntetisk oligo 25-nukleotid RNA, som indeholder en adenin-rige element (er) motiv, tillader specifikke bindende cytosole er-bindende protein.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Research

JoVE Journal

Cancer Research

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