Name: utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes
Uploaded: 2020-11-09T15:45:00
Description: Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Dette arbejde blev støttet af et FAPESP-tilskud (15/06477-9).

1. Gær total ekstrakt forberedelse
<ol><li>Knyt gærcellerne, der udtrykker en af splejsning faktorer smeltet til TAP tag9 i 1 L YNB-glu medier (Gær Nitrogen Basis suppleret med 2% m / v glukose) med de relevante aminosyrer eller nuklein baser, i dette tilfælde, adenin (20 μg/mL), leucin (30 μg/mL), tryptofan (30 μg/mL), op til OD600 = 1,0.</li>
	<li>Cellerne opsamles fra 1 L-kulturen ved at centrifugere i tre 500 mL centrifugeflasker ved 17.000 x g i 10 min ved 4 °C o...

<ol>
	<li>Kastner, B., Will, C. L., Stark, H., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+nuclear+pre-mRNA+splicing+in+higher+eukaryotes.">Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.</a> Cold Spring Harbor Perspectives in Biology. 11 (11), 032417 (2019).</li><li>Yan, C., Wan, R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+pre-mRNA+splicing+through+structural+biology+of+the+spliceosome.">Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.</a> Cold Spring Harbor Perspectives in Biology. 11 (1), 032409 (2019).</li><li>Cordin, O., Hahn, D., Beggs, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+regulation+of+spliceosomal+RNA+helicases.">Structure, function and regulation of spliceosomal RNA helicases.</a> Current Opinion in Cell Biology. 24, 431-438 (2012).</li><li>Kastner, B., 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=GraFix:+sample+preparation+for+single-particle+electron+cryomicroscopy.">GraFix: sample preparation for single-particle electron cryomicroscopy.</a> Nature Methods. 5 (1), 53-55 (2008).</li><li>Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafix:+stabilization+of+fragile+macromolecular+complexes+for+single+particle+cryo-EM.">Grafix: stabilization of fragile macromolecular complexes for single particle cryo-EM.</a> Methods in Enzymology. 481, 109-126 (2010).</li><li>Yan, C., Wan, R., Bai, R., Huang, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+yeast+activated+spliceosome+at+3.5+&#197;+resolution.">Structure of a yeast activated spliceosome at 3.5 &#197; resolution.</a> Science. 353 (6302), 895-904 (2016).</li><li>Ohi, M. 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=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+yeast+mutant+RNA+exosome+complexes.">Proteomic analysis of yeast mutant RNA exosome complexes.</a> Journal of Proteome Research. 12 (12), 5912-5922 (2013).</li><li>Rigaut, G., 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+generic+protein+purification+method+for+protein+complex+characterizatioin+and+proteome+exploration.">A generic protein purification method for protein complex characterizatioin and proteome exploration.</a> Nature Biotechnology. 17, 1030-1032 (1999).</li><li>Cheng, S. C., Newman, A., Lin, R. J., McFarland, G. D., Abelson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+fractionation+of+yeast+splicing+extract.">Preparation and fractionation of yeast splicing extract.</a> Methods in Enzymology. 181, 89-96 (1990).</li><li>Umen, J. G., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+a+U5+snRNP+protein+in+3'+splice+site+selection.">A novel role for a U5 snRNP protein in 3' splice site selection.</a> Genes &amp; Development. 9, 855-868 (1995).</li><li>Dunn, E. A., Rader, S. D., Hertel, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+whole+cell+splicing+extract.">Preparation of yeast whole cell splicing extract.</a> Spliceosomal Pre-mRNA Splicing: Methods and Protocols. Methods in Molecular Biology. 1126, 123-135 (2014).</li><li>Hill, H. D., Straka, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+using+bicinchoninic+acid+in+the+presence+of+sulfhydryl+reagents.">Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.</a> Analalytical Biochemistry. 170 (1), 203-208 (1988).</li><li>Cepeda, L. 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=The+ribosome+assembly+factor+Nop53+controls+association+of+the+RNA+exosome+with+pre-60S+particles+in+yeast.">The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.</a> The Journal of Biological Chemistry. 294 (50), 19365-19380 (2019).</li><li>Lin, R. J., Newman, A. J., Cheng, S. C., Abelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mRNA+splicing+in+vitro.">Yeast mRNA splicing in vitro.</a> The Journal of Biological Chemistry. 260 (27), 14780-14792 (1985).</li></ol>

Protein-protein og ribonukleinsyrer-protein interaktioner kan stabiliseres ved hjælp af crosslinking midler. Det er vigtigt, at det resulterende kompleks er stabilt til at modstå ultracentrifugering på glycerol gradient. Derudover bør bufferbetingelserne gøre det muligt at interagere, men være strenge nok til at undgå ikke-specifik binding. I de eksperimenter, der er vist her, brugte vi en bufferopløsning, der allerede er etableret til in vitro-splejsningsreaktioner15.
<p class="jove_c...

Splejsning er en meget dynamisk proces, der kræver binding og frigivelse af en lang række faktorer på en koordineret måde. Disse splejsning faktorer omfatter RNA bindende proteiner, ATPases, helicaser, protein kinaser og fosfattaser, allestedsnærværende ligaser, blandt andre1,2,3; og for at gøre det muligt for de molekylære omlægninger at finde sted, binder nogle af disse faktorer meget forbigående til de splejsede underkomplekser, hvilket gør isoleringen og identifikationen af disse RNP-mellemkomplekser meget udfordrende.
Her brugte vi Grafix metode4,5 til at stabilisere samspillet mellem gær splejsning faktor Cwc24 med Bhandling kompleks6 at give mulighed for identifikation af andre faktorer bundet samtidig til denne subcomplex og afgøre, om den allestedsnærværende ligase Prp19 spiller nogen rolle i bindingen eller frigivelsen af Cwc24 til Nitten (NTC) kompleks og til 5 'slutningen af intron før aktivering og den første transesterificering reaktion tager sted. Fordelen ved at udsætte makromolekylerne for en stigende koncentration af crosslinkeren langs glycerolgradienten er, at den undgår interkomplekser krydslinks4,5og dermed dannelsen af aggregater.
Denne metode blev brugt som et supplement til protein coimmunoprecipitation og pull-down assays, som, på trods af at tillade isolering af store komplekser, kan ikke være pålidelige til at opretholde forbigående interaktioner inden for store dynamiske komplekser7,8. Brugen af fiksering reagenser i glycerol gradient stabiliserer bindingen af sådanne faktorer, så bekræftelse af interaktioner af specifikke proteiner med splejsning subkomplekser. Fordi den valgte crosslinker var kemisk irreversibel, proteiner til stede i de genvundne fraktioner blev analyseret ved prik blot efter gradient centrifugation.

<table>
	<tbody>
		<tr>
			<td>Anti-Calmodulin Binding Protein Epitope</td>
			<td>Millipore</td>
			<td>07-482</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-Rabbit IgG</td>
			<td>GE Healthcare</td>
			<td>NA934</td>
			<td></td>
		</tr>
		<tr>
			<td>EconoSystem</td>
			<td>Bio-Rad</td>
			<td>1-800-424-6723</td>
			<td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td>
		</tr>
		<tr>
			<td>EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Recovery System</td>
			<td>Beckman Coulter</td>
			<td>270-331580</td>
			<td>Tube-perforating device that was connected to the parts of the EconoSystem</td>
		</tr>
		<tr>
			<td>Gradient Master Model 107ip</td>
			<td>Biocomp</td>
			<td>107-201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>207460001</td>
			<td>Ball Mill device</td>
		</tr>
		<tr>
			<td>Rotor F12-6x500Lex</td>
			<td>Thermo Scientific</td>
			<td>096-062375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>36-101-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor P40ST</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge CP 80 NX</td>
			<td>Hitachi</td>
			<td>901069</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

Pre-mRNA splejsning er en meget dynamisk proces, der involverer mange molekylære omlægninger af de splejsiske underkomplekser under montering, RNA-behandling og frigivelse af de komplekse komponenter. Glycerol gradient centrifugering er blevet anvendt til adskillelse af protein eller RNP (RiboNucleoProtein) komplekser til funktionelle og strukturelle undersøgelser. Her beskriver vi udnyttelsen af Grafix (Gradient Fiksering), som først blev udviklet til at rense og stabilisere makromolekyllære komplekser til enkelt partikel kryo-elektronmikroskopi, for at identificere interaktioner mellem splejsning faktorer, der binder forbigående til splejse kompleks. Denne metode er baseret på centrifugering af prøver i en stigende koncentration af et fikseringsreagens for at stabilisere komplekser. Efter centrifugering af gær samlede ekstrakter læsset på glycerol gradienter, genvundet fraktioner analyseres ved prik blot til identifikation af splejsning sub-komplekser og bestemmelse af tilstedeværelsen af individuelle splejsning faktorer.

For at analysere sedimenteringsprofilen af Cwc24-TAP og afgøre, om Grafix-metoden var effektiv til at stabilisere sin binding til splejsning af underkomplekser, adskilte vi samlede gærekstrakter af celler, der udtrykker Cwc24-TAP gennem centrifugering på glycerolgradienter, i nærvær eller fravær af glutaraldehyd som et krydslinkmiddel. Prøver på 24 500 μL-fraktioner blev derefter analyseret ved slot blot med antistof mod CBP-delen af TAP-mærket. Resultaterne viser , at I mangel af crosslinker er Cwc24 koncentre...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Her beskriver vi udnyttelsen af Grafix (Gradient Fiksering), en glycerol gradient centrifugering i nærværelse af en crosslinker, for at identificere interaktioner mellem splejsning faktorer, der binder forbigående til splejsning kompleks.

Udnyttelse af grafix til påvisning af forbigående interactorer af saccharomyces cerevisiae splejsning splejsstoffer

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

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Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

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