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

This work was supported by a FAPESP grant (15/06477-9).

1. Yeast total extract preparation
<ol>
	<li>Grow the yeast cells expressing one of the splicing factors fused to the TAP tag9 in 1 L YNB-glu media (Yeast Nitrogen Basis supplemented with 2% m/v glucose) with the appropriate amino acids or nucleic bases, in this case, adenine (20 &#181;g/mL), leucine (30 &#181;g/mL), tryptophan (30 &#181;g/mL), up to OD600 = 1.0.</li>
	<li>Collect the cells from the 1 L culture by centrifuging in three 500 mL centrifuge bottles at 17,000 x g ...

<ol>
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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. 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Protein-protein and ribonucleic acids-protein interactions can be stabilized using crosslinking agents. It is important that the resulting complex is stable to withstand ultracentrifugation on glycerol gradient. Additionally, the buffer conditions should allow the interaction, but be stringent enough to avoid non-specific binding. In the experiments shown here, we used a buffer solution already established for in vitro splicing reactions15.
The speed and time of centrif...

Splicing is a highly dynamic process that requires binding and release of a multitude of factors in a coordinated manner. These splicing factors include RNA binding proteins, ATPases, helicases, protein kinases and phosphatases, ubiquitin ligases, among others1,2,3; and to allow for the molecular rearrangements to take place, some of these factors bind very transiently to the spliceosome subcomplexes, making the isolation and identification of these RNP intermediate complexes very challenging.
Here, we used the Grafix method4,5 to stabilize the interaction of the yeast splicing factor Cwc24 with the Bact complex6 to allow for the identification of other factors bound concomitantly to that subcomplex and determine whether the ubiquitin ligase Prp19 plays any role in the binding or release of Cwc24 to the Nineteen (NTC) complex and to the 5&#8217; end of the intron before activation and the first transesterification reaction takes place. The advantage of exposing the macromolecules to an increasing concentration of the crosslinker along the glycerol gradient is that it avoids inter-complexes crosslinks4,5, and therefore, the formation of aggregates.
This method was used as a complementation to protein coimmunoprecipitation and pull-down assays, which, despite allowing the isolation of large complexes, may not be reliable for maintaining transient interactions within large dynamic complexes7,8. The use of fixation reagents in the glycerol gradient stabilizes the binding of such factors, allowing the confirmation of interactions of specific proteins with splicing subcomplexes. Because the chosen crosslinker was chemically irreversible, proteins present in the recovered fractions were analyzed by dot blot after the 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 splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

To analyze the sedimentation profile of Cwc24-TAP and determine whether the Grafix method was effective to stabilize its binding to splicing subcomplexes, we separated total yeast extracts of cells expressing Cwc24-TAP through centrifugation on glycerol gradients, in the presence or absence of glutaraldehyde as a crosslinking agent. Samples of twenty-four 500 &#956;L fractions were then analyzed by slot blot with antibody against the CBP portion of the TAP tag. The results show that in the absence of the crosslinker, Cwc...

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

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

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

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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