Name: a reporter based cellular assay for monitoring splicing efficiency
Uploaded: 2021-09-15T14:00:00
Description: Watch this Scientific Journal Video about A Reporter Based Cellular Assay for Monitoring Splicing Efficiency at JoVE.com

Questo lavoro è stato sostenuto da fondi a S.S. dal National Institutes of Health (R21CA170786 e R01GM127464) e dall'American Cancer Society (Institutional Research Grant 74-001-34-IRG) e a S.S. e W.M. dal Valley Research Partnership Program (P1-4009 e VRP77). Il contenuto è di esclusiva responsabilità degli autori e non rappresenta necessariamente le opinioni ufficiali del National Institutes of Health.

1. Reagenti e tamponi
NOTA: tutta la sterilizzazione con filtri a vuoto deve essere eseguita con membrana di polietersolfone (PES) da 0,2 μm in un armadio di biosicurezza.
<ol><li>Preparare acqua priva di RNasi aggiungendo 1,0 ml di dietilpirocarbonato (DEPC) a 1,0 L di acqua deionizzata, mescolare per almeno 1 ora a temperatura ambiente (RT), autoclave due volte e quindi raffreddare a RT prima dell'uso.</li>
	<li>Preparare Dulbecco's Modified Eagle Medium (DMEM) mescolando un pacchetto di ...

<ol>
	<li>Zhuang, Y., Weiner, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+compensatory+base+change+in+U1+snRNA+suppresses+a+5'+splice+site+mutation.">A compensatory base change in U1 snRNA suppresses a 5' splice site mutation.</a> Cell. 46 (6), 827-835 (1986).</li><li>Scotti, M. M., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mis-splicing+in+disease.">RNA mis-splicing in disease.</a> Nature Review Genetics. 17 (1), 19-32 (2016).</li><li>Ward, A. J., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathobiology+of+splicing.">The pathobiology of splicing.</a> Journal of Pathology. 220 (2), 152-163 (2010).</li><li>Scalet, D., 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=Disease-causing+variants+of+the+conserved++2T+of+5'+splice+sites+can+be+rescued+by+engineered+U1snRNAs.">Disease-causing variants of the conserved +2T of 5' splice sites can be rescued by engineered U1snRNAs.</a> Human Mutatation. 40 (1), 48-52 (2019).</li><li>Yamazaki, 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=Use+of+modified+U1+small+nuclear+RNA+for+rescue+from+exon+7+skipping+caused+by+5'-splice+site+mutation+of+human+cathepsin+A+gene.">Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5'-splice site mutation of human cathepsin A gene.</a> Gene. 677, 41-48 (2018).</li><li>Yanaizu, M., Sakai, K., Tosaki, Y., Kino, Y., Satoh, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+nuclear+RNA-mediated+modulation+of+splicing+reveals+a+therapeutic+strategy+for+a+TREM2+mutation+and+its+post-transcriptional+regulation.">Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation.</a> Science Reports. 8 (1), 6937 (2018).</li><li>Balestra, D., 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=Splicing+mutations+impairing+CDKL5+expression+and+activity+can+be+efficiently+rescued+by+U1snRNA-based+therapy.">Splicing mutations impairing CDKL5 expression and activity can be efficiently rescued by U1snRNA-based therapy.</a> International Journal of Molecular Sciences. 20 (17), 20174130 (2019).</li><li>Donadon, I., 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=Exon-specific+U1+snRNAs+improve+ELP1+exon+20+definition+and+rescue+ELP1+protein+expression+in+a+familial+dysautonomia+mouse+model.">Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model.</a> Human Molecular Genetics. 27 (14), 2466-2476 (2018).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> Journal of Visualized Experiments. (45), e2565 (2010).</li><li>Summer, H., Gramer, R., Droge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+urea+polyacrylamide+gel+electrophoresis+(Urea+PAGE).">Denaturing urea polyacrylamide gel electrophoresis (Urea PAGE).</a> Journal of Visualized Experiments. (32), e1485 (2009).</li><li>Dominski, Z., Kole, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+splice+sites+in+pre-mRNAs+with+short+internal+exons.">Selection of splice sites in pre-mRNAs with short internal exons.</a> Molecular Cell Biology. 11 (12), 6075-6083 (1991).</li><li>Amir-Ahmady, B., Boutz, P. L., Markovtsov, V., Phillips, M. L., Black, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-loop+4+of+U1+snRNA+is+essential+for+splicing+and+interacts+with+the+U2+snRNP-specific+SF3A1+protein+during+spliceosome+assembly.">Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly.</a> Genes and Development. 28 (22), 2518-2531 (2014).</li><li>Steitz, J. A., 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=.">.</a> Functions of the abundant U-snRNPs. Structure and function of major and minor small nuclear ribonucleoprotein particles. , 115-154 (1988).</li><li>Fortes, P., 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=Inhibiting+expression+of+specific+genes+in+mammalian+cells+with+5'+end-mutated+U1+small+nuclear+RNAs+targeted+to+terminal+exons+of+pre-mRNA.">Inhibiting expression of specific genes in mammalian cells with 5' end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA.</a> Proceedings of the National Academy of Sciences U.S.A. 100 (14), 8264-8269 (2003).</li><li>Roca, X., 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=Widespread+recognition+of+5'+splice+sites+by+noncanonical+base-pairing+to+U1+snRNA+involving+bulged+nucleotides.">Widespread recognition of 5' splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides.</a> Genes and Development. 26 (10), 1098-1109 (2012).</li><li>Roca, X., Krainer, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+atypical+5'+splice+sites+by+shifted+base-pairing+to+U1+snRNA.">Recognition of atypical 5' splice sites by shifted base-pairing to U1 snRNA.</a> Nature Structural Molecular Biology. 16 (2), 176-182 (2009).</li><li>Taladriz-Sender, A., Campbell, E., Burley, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splice-switching+small+molecules:+A+new+therapeutic+approach+to+modulate+gene+expression.">Splice-switching small molecules: A new therapeutic approach to modulate gene expression.</a> Methods. 167, 134-142 (2019).</li><li>Hamid, F. M., Makeyev, E. 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Il test può essere adattato per l'analisi di splicing in linee cellulari diverse da HeLa, tuttavia, potrebbe essere necessario ottimizzare i fattori che influenzano l'efficienza della trasfezione, come la confluenza cellulare e la quantità di DNA. Il rapporto tra il costruttore reporter e U1 è un altro parametro critico che potrebbe dover essere determinato in base ai livelli di espressione osservati in altri tipi di cellule. La qualità dell'RNA estratto è fondamentale per l'analisi dello splicing; pertanto, l'uso d...

Lo splicing pre-mRNA è una fase di elaborazione essenziale che rimuove gli introni non codificanti e lega precisamente gli esoni codificanti per formare mRNA maturo. Il riconoscimento delle sequenze di consenso alle giunzioni esone-introne, denominate sito di giunzione 5′ e sito di giunzione 3′, da parte dei componenti del macchinario di giunzione avvia il processo di giunzione. La piccola ribonucleoproteina nucleare U1 (snRNP) riconosce il sito di giunzione 5′ mediante accoppiamento di basi dello snRNA U1 al pre-mRNA1. Le mutazioni geneticamente ereditate che alterano le sequenze del sito di giunzione 5′ sono associate a molte malattie2,3. Si prevede che la perdita di basepairing di SnRNA U1 con i siti mutanti di giunzione 5′ causi uno splicing aberrante, che può compromettere la traduzione del trascritto interessato. Un potenziale approccio terapeutico per correggere i difetti di splicing comporta la soppressione delle mutazioni da parte di snRNA U1 modificato che trasporta cambiamenti nucleotidici compensatori nella sua regione 5′ che le parti base con il sito di giunzione 5′. Tali snRNA U1 modificati, noti anche come snRNA U1 specifici dell'esone, sono risultati efficaci nell'invertire i difetti di splicing, con conseguente aumento dell'espressione proteica dall'mRNA salvato4,5,6,7,8.
Qui, descriviamo il test di complementazione U1 snRNP, un test di splicing cellulare basato su reporter che consente di valutare l'effetto delle mutazioni 5′-ss sullo splicing di un esone e può anche essere utilizzato per lo sviluppo di snRNA U1 modificati per consentire il salvataggio dell'inclusione di esoni. Forniamo anche protocolli per il monitoraggio delle trascrizioni del reporter spliced tramite estensione primer e RT-PCR e per determinare l'espressione di snRNA U1 modificati mediante estensione primer e RT-qPCR.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent Grade Deionized Water</td>
			<td>ThermoFisher Scientific</td>
			<td>23-751628</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>D5758-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) powder packet</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>ThermoFisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Australian Source, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, Standard Solution 1.0N</td>
			<td>Sigma-Aldrich</td>
			<td>S2567-16A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid, Certified ACS Plus, 36.5 to 38.0%</td>
			<td>ThermoFisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Bottle Top Filters</td>
			<td>ThermoFisher Scientific</td>
			<td>FB12566510</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Disodium Salt Dihydrate</td>
			<td>Amresco</td>
			<td>0105-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10x), no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagent</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Bioreagent</td>
			<td>BP366-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium Hydrogen Phosphate Heptahydrate</td>
			<td>Fisher Bioreagent</td>
			<td>BP332-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>Fisher Bioreagent</td>
			<td>BP362-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection medium - Opti-MEM&#8482; I Reduced Serum Medium, no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Reagent - Lipofectamine&#8482; 2000</td>
			<td>ThermoFisher Scientific</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol&#8482; Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Absolute (200 Proof), Molecular Biology Grade, Fisher BioReagents</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, Molecular Biology Grade, Fisher BioReagents</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2618-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen (5 mg/ml)</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>R2052</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, [&#947;-32P]- 6000Ci/mmol 150mCi/ml Lead, 1 mCi</td>
			<td>PerkinElmer</td>
			<td>NEG035C001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201L</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion beands - Sephadex&#174; G-25</td>
			<td>Sigma-Aldrich</td>
			<td>G2580-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion mini columns</td>
			<td>USA Scientific</td>
			<td>1415-0600</td>
			<td></td>
		</tr>
		<tr>
			<td>pBR322 DNA-MspI Digest</td>
			<td>New England Biolabs</td>
			<td>N3032S</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Molecular Weight Marker, 10-100 nt</td>
			<td>Affymetrix</td>
			<td>76410 100 UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase inactivating reagents - RNaseZAP&#8482;</td>
			<td>Sigma-Aldrich</td>
			<td>R2020-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix (10 mM ea)</td>
			<td>ThermoFisher Scientific</td>
			<td>18427013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseOUT&#8482; Recombinant Ribonuclease Inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcriptase - M-MLV Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>28025013</td>
			<td>used for primer extension</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>10342020</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Hexamers (50 &#181;M)</td>
			<td>ThermoFisher Scientific</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time PCR mix - SYBR&#8482; Select Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>4472903</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript&#8482; III Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td>used for cDNA preparation</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td></td>
		</tr>
		<tr>
			<td>5X First-Strand Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>18080093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide (&#8805;99.5%)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP228-100</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Bromophenol Blue sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>114405-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene Cyanol FF</td>
			<td>Sigma-Aldrich</td>
			<td>2650-17-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base (White Crystals or Crystalline Powder/Molecular Biology)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid (Crystalline/Electrophoresis)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP168-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide: Bis-Acrylamide 19:1 (40% Solution/Electrophoresis)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1406-1</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Urea (Colorless-to-White Crystals or Crystalline Powder/Mol. Biol.)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP169-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium peroxodisulphate (APS) &#8805;98%, Pro-Pure, Proteomics Grade</td>
			<td>VWR</td>
			<td>M133-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED) &#8805;99%, Ultrapure</td>
			<td>VWR</td>
			<td>0761-25ML</td>
			<td>Review Material Safety Data Sheets</td>
		</tr>
		<tr>
			<td>Adjustable Slab Gel Systems, Expedeon</td>
			<td>VWR</td>
			<td>ASG-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 14.5cm</td>
			<td>VWR</td>
			<td>NGP-125NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 22.0cm</td>
			<td>VWR</td>
			<td>NGP-200NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Gel Wrap&#8482; Glass Plate Sets, 16.5 x 38.7cm</td>
			<td>VWR</td>
			<td>NGP-400NR</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage Phosphor Screens</td>
			<td>Sigma-Aldrich</td>
			<td>GE28-9564</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon&#8482; FLA 7000 Biomolecular Imager</td>
			<td>GE Healthcare</td>
			<td>28-9610-73 AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter LS6500 Liquid Scintillation Counter</td>
			<td>GMI</td>
			<td>8043-30-1194</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 6 Flex Real-Time PCR Systems</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a reporter based cellular assay for monitoring splicing efficiency

Durante l'espressione genica, la fase vitale dello splicing pre-mRNA comporta un riconoscimento accurato dei siti di giunzione e un assemblaggio efficiente di complessi spliceosomiali per unire gli esoni e rimuovere gli introni prima dell'esportazione citoplasmatica dell'mRNA maturo. L'efficienza dello splicing può essere alterata dalla presenza di mutazioni nei siti di giunzione, dall'influenza di fattori di splicing trans-agenti o dall'attività terapeutica. Qui, descriviamo il protocollo per un test cellulare che può essere applicato per monitorare l'efficienza di giunzione di un dato esone. Il test utilizza un reporter minigene plasmidico codificato plasmidico 3-esone/2-introne adattabile, che può essere espresso in cellule di mammifero mediante trasfezione transitoria. Dopo la trasfezione, l'RNA cellulare totale viene isolato e l'efficienza dello splicing dell'esone nell'mRNA reporter è determinata dall'estensione del primer o dalla reazione a catena semi-quantitativa della trascrittasi-polimerasi inversa (RT-PCR). Descriviamo come l'impatto delle mutazioni del sito di giunzione 5′ associate alla malattia può essere determinato introducendole nel reporter; e come la soppressione di queste mutazioni può essere ottenuta mediante co-trasfezione con il piccolo costrutto di RNA nucleare U1 (snRNA) che porta mutazioni compensatorie nella sua regione 5′ che le parti base con i siti di giunzione 5′-giunzione alle giunzioni esone-introne nei pre-mRNA. Pertanto, il reporter può essere utilizzato per la progettazione di particelle U1 terapeutiche per migliorare il riconoscimento dei siti di giunzione 5′ mutanti. L'inserimento di siti regolatori ad azione cis, come il potenziatore di splicing o le sequenze di silenziatori, nel reporter può anche essere utilizzato per esaminare il ruolo dell'U1 snRNP nella regolazione mediata da uno specifico fattore di splicing alternativo. Infine, le cellule che esprimono reporter possono essere incubate con piccole molecole per determinare l'effetto di potenziali terapie sullo splicing costitutivo pre-mRNA o su esoni portatori di siti di giunzione 5′ mutanti. Nel complesso, il test reporter può essere applicato per monitorare l'efficienza dello splicing in una varietà di condizioni per studiare i meccanismi fondamentali di splicing e le malattie associate allo splicing.

Il reporter di splicing Dup51, un minigene di tre esoni-due introni, è stato derivato dal gene umano β-globina ed è stato descritto in precedenza (Figura 1A) 11,12 . Abbiamo creato un reporter mutante, Dup51p, introducendo mutazioni del sito di giunzione associate alla sindrome di Usher che si verificano nell'esone 3 del gene protocadherina 15 (PCDH15)13. La sequenza del sito di giunzione 5'-giunzi...

Watch this Scientific Journal Video about A Reporter Based Cellular Assay for Monitoring Splicing Efficiency at JoVE.com

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Questo protocollo descrive un minigene reporter assay per monitorare l'impatto delle mutazioni del sito di giunzione 5'-splicing sullo splicing e sviluppa snRNA U1 soppressore per il salvataggio dell'inibizione dello splicing indotta da mutazioni. I costrutti di snRNA U1 reporter e soppressore sono espressi in cellule HeLa e lo splicing viene analizzato mediante estensione di primer o RT-PCR.

Un test cellulare basato su reporter per il monitoraggio dell'efficienza di giunzione

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Questo protocollo descrive un test cellulare per il monitoraggio dell'efficienza di splicing, utilizzando un reporter di gestione adattabile. Questo reporter Assay può essere utilizzato per studiare gli effetti delle mutazioni associate alla malattia nei siti Exxon o di giunzione e per progettare la terapia, in particolare una particella, per migliorare il riconoscimento dei siti di giunzione mutanti a cinque punti. Il passaggio nelle cellule Hela aspira il mezzo esaurito e incuba le cellule con tre millilitri di 0,2 viaggi del 5% in contenenti un EDTA millimolare, a 37 gradi Celsius per tre minuti.Dopo l'incubazione a sette millilitri di DMEM fresco e trasferire la sospensione cellulare in un tubo da 10 millilitri. Centrifugare le cellule. Quindi risospendere il pellet cellulare in 10 millilitri di DMEM fresco e placcare le cellule su un nuovo piatto di coltura tissutale al 20% di confluenza, per la trasfezione transitoria.Contare le cellule hela con un vetrino emocitometrico pulito e preparare una sospensione con una densità di 2,5 volte 10 alla quinta cellula per millilitro, seminare un millilitro della sospensione cellulare in ciascun pozzetto di una piastra da 12 pozzetti e incubare durante la notte a 37 gradi Celsius. Il giorno seguente aspirare il mezzo esaurito e aggiungere 800 microlitri di DMEM fresco con siero al piatto. Preparare le miscele di trasfezione e vortexle per 15 secondi.Quindi centrifugare le miscele e incubarle a temperatura ambiente per cinque minuti. Dopo l'incubazione aggiungere tutti i 200 microlitri della miscela di trasfezione in un pozzetto della piastra di cellule hela a 12 pozzetti per ottenere un volume finale di un millilitro per pozzetto e incubare la piastra a 37 gradi Celsius per 48 ore. Le reazioni di etichettatura preparate sono in incubazione.Sospendere le perle di esclusione delle dimensioni di taglio del peso molecolare Dalton da 25 chili con un delicato vortice per 10 secondi. Successivamente trasferire 600 microlitri delle perline risospese su una mini colonna usa e getta, collocata in un tubo di raccolta da 1,5 millilitri, e riportare lo stock di perline a quattro gradi Celsius. Centrifugare la colonna e scartare il flusso attraverso di esse.Quindi lavare le perline due volte aggiungendo 300 microlitri di acqua priva di RNA alla colonna. Dopo il secondo lavaggio, trasferire la mini colonna in un nuovo tubo centrifuga da 1,5 millilitri e aggiungere la miscela di reazione chinasica alla colonna. Centrifugare la colonna e raccogliere il flusso attraverso la presenza degli oligonucleotidi marcati P32.Aggiungere due microgrammi di RNA estratto dalle elicellule in tubi microcentrifughe da 200 microlitri e aggiungere acqua priva di RNA per aumentare il volume a 6,55 microlitri. Quindi aggiungere 0,9 microlitri, un master mix uno per ogni tubo PCR contenente i campioni di RNA e incubare i tubi prima a 65 gradi Celsius per cinque minuti, quindi sul ghiaccio per un minuto. Successivamente a 2,55 microlitri, un master mix due, per ogni tubo contenente l'RNA e master mix uno, e incubare i tubi a temperatura ambiente per 10 minuti.Dopo l'incubazione trasferire i tubi a un termociclatore e incubare prima a 50 gradi Celsius per 60 minuti, e poi a 70 gradi Celsius, per 15 minuti. Dopo l'incubazione, aggiungere 10 microlitri di colorante di caricamento 2X formamide RNA a ciascun campione e procedere a separare i frammenti per pagina UIA. Sintesi del DNA di Percy.Aggiungere due microgrammi di RNA estratti dalle cellule hela transettate, in tubi microncentrifuga da 200 microlitri e aggiungere acqua priva di RNA per aumentare il volume a un basso 11 microlitri. Quindi, aggiungere due microlitri, un master mix uno per ogni campione e incubare prima a 65 gradi Celsius per cinque minuti, quindi sul ghiaccio per un minuto. Quindi, aggiungere sette microlitri, un master mix due per ogni tubo e incubare i tubi a temperatura ambiente per 10 minuti, seguiti da incubazione a 50 gradi Celsius per 60 minuti e 70 gradi Celsius per 15 minuti.Successivamente, per la PCR diluire la reazione del DNA C per produrre una concentrazione di 100 nanogrammi per microlitro e trasferire un microlitro di ciascun campione CDNA a nuovi tubi PCR. Aggiungere 10,5 microlitri del mix master a ciascun tubo contenente CDNA ed eseguire la PCR. Utilizzo di un termociclatore Al termine della PCR.Aggiungere 12,5 microlitri di colorante di caricamento del DNA 2X di formammide a ciascun tubo. Riscaldare i campioni a 95 gradi Celsius per cinque minuti e procedere con l'esecuzione di una pagina UIA. Pipettare cinque microlitri di CDNA diluito in singoli pozzi di un triplicato di piastra qpcr a 96 pozzetti.Quindi aggiungere 10 microlitri di miscela PCR in tempo reale a ciascun pozzo, sigillare le piastre con un film ottico e centrifugare per raccogliere le reazioni sul fondo dei pozzi, quindi eseguire il qpcr mentre si raccolgono i valori del ciclo di quantificazione di soglia dell'amplicon target. Prima di caricare i marcatori e i campioni, sciacquare i pozzetti con il tampone TBE per rimuovere l'urea stabilizzata. Quindi caricare 10 microlitri di ciascun campione per pozzetto ed eseguire il gel a 300-500 volt per due o tre ore o fino a quando lo xilene raggiunge il fondo.Dopo l'elettroforesi rimuovere le lastre di vetro dall'apparecchio di elettroforesi e separare accuratamente le due piastre in modo che il gel si trovi piatto su entrambe le superfici di vetro, quindi trasferire il gel su una carta da filtro e coprirlo con un involucro di plastica, utilizzando un essiccatore di gel asciugare sottovuoto il gel a 80 gradi Celsius per 30 minuti, quindi posizionare il gel essiccato in una cassetta di imaging al fosforo per l'incubazione notturna a temperatura ambiente. Quando espresso in celle hela, il reporter di affettamento dup 51 minnijean esprime la trascrizione matura a figura intera in cui Exxon due è giuntato in modo efficiente. Le cinque mutazioni principali del sito di giunzione e dup 51 P riducono l'inclusione di Exxon due dal 95 al 30%, portando alla formazione di un trascritto più breve.La co-espressione di dup 51 P con U15A SNRNA salva Exxon due splicing e ripristina la formazione del trascritto a lunghezza intera. Al contrario, la co-espressione con la variante wild type U1 o U15G non ha l'effetto simile sullo splicing dup 51P. Il rilevamento di trascritti di varianti U15A, per estensione primaria, utilizza l'oligonunzido U1 da sette a 26, che è lungo 20 nucleotidi e coppie di basi vicino all'adenina nella quinta posizione di U1 SNRNA.In presenza del solo DATP, U1 da sette a 26 consente di incorporare da due a tre nucleotidi per l'SNRNA U1 di tipo selvaggio endogeno producendo un prodotto lungo 22 o 23 nucleotidi. Per le varianti U15A e U15G l'estensione termina dopo l'aggiunta di una singola adenosina che produce un prodotto a 21 nucleotidi. Dopo la normalizzazione all'espressione di U2 SNRNA, le varianti U1 5g e U15A trasfettate di tipo U1 wild-type sono state espresse da tre a cinque volte rispetto allo SNRNA endogeno.La qualità dell'RNA estratto è fondamentale per l'analisi dello splicing. Pertanto, l'uso di acqua priva di RNA e la decontaminazione dei servizi con agenti inattivanti RNA è altamente raccomandato. Il sistema di report qui descritto può essere applicato per studiare il ruolo degli RNA SN U1 nei regolamenti di splicing per invertire l'effetto delle mutazioni di splicing del prezzo flash per il silenziamento genico utilizzando RNA U1SN modificati.

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SCIENTISTS IN ACTION

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Un test fluorescenza a base di fosfolipidi Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Ricerca

Biochimica

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Elettrochimico rilevamento di deuterio isotopo effetto cinetico su extracellulare di trasporto dell'elettrone nella Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Un test In Vitro per rilevare attività di tRNA-Isopentenyl Transferase

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Un'analisi di Luciferase High-Throughput per valutare la proteolisi di PCSK9 la proteasi singolo-fatturato

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Screening per fitoestrogeni utilizzando un recettore degli estrogeni a base cellulare β test reporter

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

Un saggio basato sull'elettrochemiluminescenza per le varianti proteiche MeCP2

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Valutazione dell'ossidazione cellulare utilizzando una proteina fluorescente verde sensibile al comparto subcellulare

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Complementazione dell'attività di splicing da parte di un complesso snRNP Galectin-3 - U1 su perline

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Screening dei frammenti basato su NMR in un campione minimo ma in modalità di automazione massima

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.