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. <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. 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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, ...

The gradient fixation method, in which a glycerol gradient centrifugation is performed in the presence of a cross-linker, helps to identify interactions between proteins that bind transiently to multi-subunit complexes. The use of fixation reagents in the glycerol gradient stabilize the bind of loose factors, without the formation of precipitates, allowing the identification of the interactions of specific proteins with spliced semi complex's. The demonstration of end protocol helps first time users to learn information that may be difficult to understand from written text alone.For yeast total extract preparation, grow the yeast cells expressing one of the splicing factors fused to the tap tag and one liter of Y and B glucose medium supplemented with the appropriate amino acids or nucleic bases. When the culture reaches an optical density at 600 nanometers of one, collect the yeast cells by centrifugation in three 500 milliliter centrifuge bottles, and wash the cells two times in 10 milliliters of cold sterile water per wash. After the second wash, resuspend the cells in one 10th of the cell volume of cold buffer A, and freeze 50 microliter drops of the yeast cell suspension in liquid nitrogen.After freezing, grind the frozen cell pellets through six cycles at 20 hertz for three minutes in a ball mill device, immersing the container of frozen cells in liquid nitrogen at the end of each cycle. After the last cycle, place the tubes containing the extracts into water at room temperature with occasional shaking. Once melted, centrifuge to collect the extracts.And quantify the protein that the cleared supernatants by the BCA method. Then fast freeze aliquots of the extracts in liquid nitrogen for minus 80 degrees Celsius storage. To prepare a glycerol gradient, add a 0.1%final concentration of the cross-linking agent glutaraldehyde to a 30%glycerol and buffer A solution, and mixed to homogenize.Add six milliliters of cold 10%glycerol and buffer A solution to a 12 milliliter centrifuge tube and use a gradient master device syringe equipped with a long needle to layer the 30%glycerol glutaraldehyde solution at the bottom of the tube. Use a gradient master device to create a linear glycerol gradient according to the manufacturer's specifications. Carefully layer 200 microliters of a 7%glycerol and buffer A cushion to the top of the gradient before adding approximately two milligrams of cell extract to the top of the tube.After glycerol gradient generation, place the sample in a pre cooled swing bucket rotor and collect the extract by centrifugation. Aliquot the sample from each 12 milliliter sample tube in 24 500 microliter fractions, and use a peristaltic pump to push a 40%glycerol solution through the 10 to 30%gradient from the tube to the fraction collection. Then load 50 microliters of each fraction directly onto the nitrocellulose membranes on a dot blot, followed by addition of blocking buffer, and use a primary antibody against CBP to detect the protein on the immuno blot, and an appropriate secondary antibody as necessary.In the absence of cross-linker CWC 24 is concentrated between fractions five and 13, corresponding to complexes of about 80 to 200 megadaltons. In the presence of the cross-linker, however, the position of CWC 24 on the gradient is shifted to fractions at the bottom of the gradient corresponding to larger complexes. Comparing CWC 24 sedimentation to that of the U5 snRNP subunit, Prp8, and the NTC subunit, Prp19 reveals that CWC 24 is concentrated in the same fractions.But, because it associates transiently with the spliceosome, it is also present in the lighter fractions. Interestingly, fractions 11 and 12 are those in which Prp8 and 19 start to concentrate, suggesting that this is the portion of the gradient where the B Act complex begins to sediment. Western blot analysis of fractions 11 and 12 reveals Prp8 and 19 signals as smears that barely enter an 8%acrylamide gel, showing that they are indeed part of large complexes.CWC 24 is also present in fraction 12, but in a much lower concentration, consistent with its transient binding to the B Act complex. Taken together, these results show that glutaraldehyde can be used as a cross-linker to stabilize the binding of transient factors to splicing sub complexes. This method can be applied to the study of any dynamic motor sub-unit complexes that associates transiently with some of their components, such as the spliceosome.

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Biochemistry

3.4K ビュー.  University of Sao Paulo. The gradient fixation method, in which a glycerol gradient centrifugation is performed in the presence of a cross-linker, helps to identify interactions between proteins that bind transiently to multi-subunit complexes. The use of fixation reagents in the glycerol gradient stabilize the bind of loose factors, without the formation of precipitates, allowing the identification of the interactions of specific proteins with spliced semi complex's. The demonstration of end protocol helps first tim...