This work has been supported by National Science Foundation Grant MCB-0092919 and Michigan State University Intramural Research Grant 09-CDFP-2001 (to RJP) and by National Institutes of Health Grant GM-38740 and Michigan AgBioResearch Project MICL02455 (to JLW).
The MINX pre-mRNA substrate used in the splicing assays was a kind gift from Dr. Susan Berget (Baylor College of Medicine, Houston, TX, USA).

1. Notes on general procedures
<ol>
	<li>Ensure that all chemicals (buffer components, enzymes, etc.) are kept free of ribonuclease (RNase). Sequester all commercially purchased reagent bottles from general lab use. Wear gloves for all steps of the experimental procedure. Use only glassware and utensils that have been baked (see step 1.2 below) and solutions that have been pre-treated (see step 1.3 below).</li>
	<li>Bake all glassware (beakers, flasks, bottles, pipettes, etc.) for a minimum of 4 h at 177 &#176;C. Wrap ...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snurps+and+scyrps.">Snurps and scyrps.</a> Cell. 25, 298-300 (1981).</li><li>Maniatis, T., Reed, 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+small+nuclear+ribonucleoprotein+particles+in+pre-mRNA+splicing.">The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing.</a> Nature. 325, 673-678 (1987).</li><li>Hoskins, A. 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=Ordered+and+dynamic+assembly+of+single+spliceosomes.">Ordered and dynamic assembly of single spliceosomes.</a> Science. 331, 1289-1295 (2011).</li><li>Coppin, L., Leclerc, J., Vincent, A., Porchet, N., Pigny, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Messenger+RNA+life-cycle+in+cancer:+emerging+role+of+conventional+and+non-conventional+RNA-binding+proteins.">Messenger RNA life-cycle in cancer: emerging role of conventional and non-conventional RNA-binding proteins.</a> International Journal of Molecular Sciences. 19, 650-676 (2018).</li><li>Dagher, S. F., Wang, J. L., Patterson, R. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+spliceosomal+RNAs+by+antisera+to+galectin-1+and+galectin-3.">Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3.</a> Nucleic Acids Research. 34, 5166-5174 (2006).</li><li>Haudek, K. C., Voss, P. G., Locascio, L. E., Wang, J. L., Patterson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+incorporation+of+galectin-3+into+the+spliceosome+through+its+association+with+U1+snRNP.">A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP.</a> Biochemistry. 48, 7705-7712 (2009).</li><li>Fritsch, K., 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=Galectin-3+interacts+with+components+of+the+nuclear+ribonucleoprotein+complex.">Galectin-3 interacts with components of the nuclear ribonucleoprotein complex.</a> BMC Cancer. 16, 502-511 (2016).</li><li>Conway, G. C., Krainer, A. R., Spector, D. L., Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+splicing+factors+are+released+from+endogenous+complexes+during+in+vitro+pre-mRNA+splicing.">Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.</a> Molecular and Cellular Biology. 9, 5273-5280 (1989).</li><li>Dery, K. J., Yean, S. L., Lin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+glycerol+gradient+isolation+of+yeast+spliceosomes+containing+transcribed+or+synthetic+U6+snRNA.">Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.</a> Methods in Molecular Biology. 488, 41-63 (2008).</li><li>Yoshimoto, R., Kataoka, N., Okawa, K., Ohno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+post-splicing+lariat-intron+complexes.">Isolation and characterization of post-splicing lariat-intron complexes.</a> Nucleic Acids Research. 37, 891-902 (2009).</li><li>Malca, H., Shomron, N., Ast, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+U1+snRNP+base+pairs+with+the+5'+splice+site+within+a+penta-snRNP+complex.">The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex.</a> Molecular and Cellular Biology. 23, 3442-3455 (2003).</li><li>Haudek, K. C., Voss, P. G., Wang, J. L., Patterson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+10S+galectin-3+-+snRNP+complex+assembles+into+active+spliceosomes.">A 10S galectin-3 - snRNP complex assembles into active spliceosomes.</a> Nucleic Acids Research. 44, 6391-6397 (2016).</li><li>Rappsilber, J., Ryder, U., Lamond, A. I., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+proteomic+analysis+of+the+human+spliceosome.">Large-scale proteomic analysis of the human spliceosome.</a> Genome Research. 12, 1231-1245 (2002).</li><li>Jurica, M. S., Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+splicing+complexes+to+study+structure+and+mechanism.">Capturing splicing complexes to study structure and mechanism.</a> Methods. 28, 336-345 (2002).</li><li>Patterson, R. J., Haudek, K. C., Voss, P. G., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+the+role+of+galectins+in+pre-mRNA+splicing.">Examination of the role of galectins in pre-mRNA splicing.</a> Methods in Molecular Biology. 1207, 431-449 (2015).</li><li>Dignam, J. D., Lebovitz, R. M., Roeder, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+transcription+initiation+by+RNA+polymerase+II+in+a+soluble+extract+from+isolated+mammalian+nuclei.">Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.</a> Nucleic Acids Research. 11, 1475-1489 (1983).</li><li>Agarwal, N., Sun, Q., Wang, S. Y., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate-binding+protein+35.+I.+Properties+of+the+recombinant+polypeptide+and+the+individuality+of+the+domains.">Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.</a> Journal of Biological Chemistry. 268, 14932 (1993).</li><li>Zillmann, M., Zapp, M. I., Berget, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+electrophoretic+isolation+of+splicing+complexes+containing+U1+small+nuclear+ribonucleoprotein+particles.">Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.</a> Molecular and Cellular Biology. 8, 814-821 (1988).</li><li>Barondes, S. H., 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=Galectins:+a+family+of+animal+&#946;-galactoside-binding+proteins.">Galectins: a family of animal &#946;-galactoside-binding proteins.</a> Cell. 76, 597-598 (1994).</li><li>Laing, J. G., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+carbohydrate+binding+protein+35+in+heterogeneous+nuclear+ribonucleoprotein+complex.">Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex.</a> Biochemistry. 27, 5329-5334 (1988).</li><li>Vyakarnam, A., Lenneman, A. J., Lakkides, K. M., Patterson, R. J., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+nuclear+localization+study+of+galectin-1+with+other+splicing+components.">A comparative nuclear localization study of galectin-1 with other splicing components.</a> Experimental Cell Research. 242, 419-428 (1998).</li><li>Michaud, S., Reed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ATP-independent+complex+commits+pre-mRNA+to+the+mammalian+spliceosome+assembly+pathway.">An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway.</a> Genes &amp; Development. 5, 2534-2546 (1991).</li><li>Chiu, Y. -. F., 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=Cwc25+is+a+novel+splicing+factor+required+after+Prp2+and+Yju2+to+facilitate+the+first+catalytic+reaction.">Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.</a> Molecular and Cellular Biology. 29, 5671-5678 (2009).</li><li>Krishnan, R., 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=Biased+Brownian+ratcheting+leads+to+pre-mRNA+remodeling+and+capture+prior+to+first-step+splicing.">Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.</a> Nature Structural and Molecular Biology. 20, 1450-1457 (2013).</li><li>Gray, R. M., 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=Distinct+effects+on+splicing+of+two+monoclonal+antibodies+directed+against+the+amino-terminal+domain+of+galectin-3.">Distinct effects on splicing of two monoclonal antibodies directed against the amino-terminal domain of galectin-3.</a> Archives of Biochemistry and Biophysics. 475, 100-108 (2008).</li></ol>

This report provides the experimental details that document a Gal3 - U1 snRNP complex trapped on anti-Gal3 coated beads can bind to pre-mRNA substrate and this ternary complex can restore splicing activity to an U1 snRNP-depleted NE. Gal3 is one member of a family of proteins originally isolated on the basis of its galactose-specific carbohydrate-binding activity23. Early immunofluorescence and subcellular fractionation studies provided the initial hint of an association of Gal3 with components of...

Production of most eukaryotic messenger RNAs (mRNAs) involves removal of introns and ligation of exons in a nuclear process termed pre-mRNA splicing1. Two classes of RNA-protein complexes (RNPs) direct the processing of pre-messenger RNA into mature mRNA via spliceosomal complexes. One class, nascent pre-messenger RNPs, is formed co-transcriptionally by the binding of heterogeneous nuclear RNP proteins and other RNA-binding proteins, including some members of the SR family, yielding hnRNP complexes2. The second class, uracil-rich small nuclear RNPs (U snRNPs with U1, U2, U4, U5, and U6 snRNAs) is associated with U-specific and core proteins3,4. The U snRNPs interact in an ordered fashion with specific regions of pre-messenger RNPs in a dynamic remodeling pathway as introns are excised and exons are ligated to produce mature mRNPs5. Many additional nuclear proteins participate in these processing events6.
Galectin-1 (Gal1) and galectin-3 (Gal3) are two proteins that are required factors in the splicing pathway as shown by depletion-reconstitution studies7,8. Removal of both galectins from splicing competent nuclear extracts (NE) abolishes spliceosome assembly and splicing activity at an early step. Addition of either galectin to such a doubly depleted NE restores both activities. Gal1 and Gal3 are components of active spliceosomes as evidenced by specific immunoprecipitation of pre-mRNA, splicing intermediates, and mature mRNA by antiserum specific for either Gal1 or Gal39. Importantly, Gal3 associates with endogenous U snRNA containing particles in the NE outside the splicing pathway as shown by precipitation of snRNPs by anti-Gal3 antisera10. Finally, silencing of Gal3 in HeLa cells alters splicing patterns of numerous genes11.
In NE pre-incubated to disassemble preformed spliceosomes12, snRNPs are found in multiple complexes sedimenting in glycerol gradients from 7S to greater than 60S. Although glycerol gradient fractionation is a common technique for the isolation of spliceosomal complexes and components (see references13,14,15 for example), we have extended this method by characterizing specific fractions using antibody immunoprecipitations. An snRNP sedimenting at 10S contains only U1 snRNA along with Gal3. Immunoprecipitation of the 10S fraction with antisera specific for Gal3 or U1 snRNP co-precipitates both U1 and Gal3 indicating some of the U1 snRNP monoparticles are bound to Gal310. As U1 snRNP is the first complex that binds to pre-mRNP in spliceosomal assembly1,5, this step represents a potential entry site for Gal3 into the splicing pathway. On this basis, we showed that 10S Gal3-U1 snRNP monoparticles bound to anti-Gal3 containing beads restored splicing activity to a U1 snRNP depleted NE, establishing this complex as one mechanism by which Gal3 is recruited into the spliceosomal pathway16. This contrasts with attempts to isolate spliceosomes at specific stages in the splicing reaction and cataloging the associated factors17,18. In such studies, the presence of certain factors at some time point is ascertained but not the mechanism by which they were loaded.
We had previously described in detail the preparation of NE, the splicing substrate, the assembly of the splicing reaction mixture, and the analysis of products in our documentation of the role of galectins in pre-mRNA splicing19. We now describe the experimental procedures for fractionation of nuclear extracts to obtain a fraction enriched in Gal3 - U1 snRNP complex and for immuno-selection of the latter complex to reconstitute splicing activity in a U1-depleted nuclear extract.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61990/61990fig01.jpg" /> 
Figure 1: Schematic diagram illustrating the complementation of splicing activity in nuclear extract depleted of U1 snRNP by a Gal3-U1 snRNP complex on beads. (A) NE in Buffer C (NE(C)) is incubated with Protein A-Sepharose beads covalently coupled with anti-U1 snRNP (&#945;U1 beads). The unbound fraction is depleted of U1 snRNP (U1&#916;NE). (B) NE in Buffer D (NE(D)) is fractionated over a 12%-32% glycerol gradient by ultracentrifugation. Fractions corresponding to the 10S region (fractions 3-5) are combined and mixed with beads covalently coupled with anti-Gal3 antibodies (&#945;Gal3 beads). The material bound to the beads contains a Gal3-U1 snRNP monoparticle. (C) The Gal3-U1 snRNP complex from Part (B) is mixed with U1&#916;NE from Part (A) in a splicing assay using&#160;32P-labeled MINX pre-mRNA substrate and the intermediates and products of the splicing reaction are analyzed by gel electrophoresis and autoradiography.&#160;<a href="https://www.jove.com/files/ftp_upload/61990/61990fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-U1 snRNP</td>
			<td>The Binding Site</td>
			<td>Hu ENA-RNP #33471</td>
			<td>human autoimmune serum specific for U1 snRNP</td>
		</tr>
		<tr>
			<td>bottle top vacuum filter</td>
			<td>Fisher Scientific</td>
			<td>Corning 431153 (0.22 &#956;m; PES 150 ml)</td>
			<td>for filtering solutions containing Tris</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>International Equipment Company</td>
			<td>IEC Model PR-6</td>
			<td>for pelletting Sepharose beads in immunoprecipitation</td>
		</tr>
		<tr>
			<td>diethylpyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>159220-5G</td>
			<td>for treatment of water used in preparation of all solutions</td>
		</tr>
		<tr>
			<td>dimethylpimelimidate (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>80490-5G</td>
			<td>for cross-linking antibody to Sepharose beads</td>
		</tr>
		<tr>
			<td>electrophoresis cell</td>
			<td>BioRad Laboratories, Inc</td>
			<td>Mini-Protean II</td>
			<td>for SDS-PAGE separation of proteins</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>411000-100ml</td>
			<td>for blocking after the cross-linking reaction</td>
		</tr>
		<tr>
			<td>gel electrophoresis system</td>
			<td>Hoefer, Inc</td>
			<td>HSI SE 500 Series</td>
			<td>for separating snRNAs by gel electrophoresis</td>
		</tr>
		<tr>
			<td>gel slab dryer</td>
			<td>BioRad</td>
			<td>Model 224</td>
			<td>for drying gel slabs for autoradiography</td>
		</tr>
		<tr>
			<td>Hybond ECL membrane</td>
			<td>GE Healthcare</td>
			<td>RPN3032D (0.2 &#956;m; 30 cm x 3 m)</td>
			<td>for immunoblotting of proteins on membrane</td>
		</tr>
		<tr>
			<td>microdialyzer (12 x 100 &#956;l sample capacity)</td>
			<td>Pierce</td>
			<td>Microdialyzer System 100</td>
			<td>for exchanging the buffer of nuclear extract&#160;&#160;</td>
		</tr>
		<tr>
			<td>microdialyzer membranes (8K cutoff)</td>
			<td>Pierce</td>
			<td>66310</td>
			<td>for exchanging the buffer of nuclear extract&#160;</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Spartan Stores</td>
			<td>Spartan Instant Non-fat Dry Milk</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A Sepharose CL-4B</td>
			<td>Millipore-Sigma</td>
			<td>GE 17-0780-01</td>
			<td>for coupling antibody to beads</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Millipore-Sigma</td>
			<td>P2308-5mg</td>
			<td>for stopping the splicing reaction to isolate the RNAs</td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N2111</td>
			<td>for inhibiting ribonuclease activity</td>
		</tr>
		<tr>
			<td>rocker/rotator</td>
			<td>Lab Industries, Inc&#160;</td>
			<td>Labquake Shaker 400-110</td>
			<td>for mixing protein solutions in coupling reactions and in immunoprecipitation</td>
		</tr>
		<tr>
			<td>Safety-Solve</td>
			<td>Research Products International Corp.</td>
			<td>No. 111177</td>
			<td>scintillation counting cocktail for determination of radioactivity in splicing substrate</td>
		</tr>
		<tr>
			<td>scintillation counter</td>
			<td>Beckman Instruments</td>
			<td>LS6000SC</td>
			<td>scintillation counter for determination of radioactivity&#160;</td>
		</tr>
		<tr>
			<td>speed vaccum concentrator</td>
			<td>Savant</td>
			<td>SVC 100H</td>
			<td>for drying ethanol-precipitated RNA pellets</td>
		</tr>
		<tr>
			<td>Transphor electrophoresis unit</td>
			<td>Hoefer, Inc</td>
			<td>Hoefer TE Series Transphor</td>
			<td>for protein transfer from SDS-PAGE to blotting membrane</td>
		</tr>
	</tbody>
</table>

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.

NE depleted of U1 snRNP (U1&#916;NE from Section 2.2.6) and Gal3 - U1 snRNP complexes from the 10S region of the glycerol gradient immunoprecipitated by anti-Gal3 (step 3.2.7) were mixed in a splicing reaction.&#160;This reaction mixture contained U1 snRNA (Figure 2A, lane 3), as well as the U1-specific protein, U1-70K (Figure 2B, lane 3). As expected, the anti-Gal3 precipitated Gal3 (Figure 2B, lane...

Watch this Scientific Journal Video about Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads at JoVE.com

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

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.

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

This report provides the experimental evidence for the documentation that a galectin-3 U1 snRNP complex binds to pre mRNA substrate forms a functional complex and leads to products of the splicing reaction. This protocol utilizes the galectin-3 U1 snRNP immunoselected on beads covalently coupled to anti-gal3 antibodies to initiate the splicing reaction, which can progress to completion. A critical step in the protocol is the careful removal of liquid after pelleting the beads containing the galectin-3 U1 snRNP complex and its immediate use in initiating the splicing reaction.To deplete U1 snRNPs from the nuclear extract, incubate 200 microliters of nuclear extract with 100 microliters of anti-U1 beads. Add five microliters of RNAs into the mixture and rotate the micro tube head over tail at four degrees Celsius for one hour. Pellet the mixture by centrifugation and collect the unbound material using a Hamilton syringe.Dialyze the entire volume of U1-depleted nuclear extract along with a separate 50 microliter aliquot of the original non-depleted nuclear extract in separate compartments of a micro dialyzer with stirring for 75 minutes against 60%buffer D at four degrees Celsius. Use a dialysis membrane with an 8K molecular weight cut off. Immediately after dialysis, divide the preparations into 20 microliter aliquots, then snap freeze them in a dry ice ethanol bath and store them at minus 80 degrees Celsius.Just before use, wash the anti-gal3 beads twice with 0.5 milliliters of TX wash buffer. For each wash, add the wash buffer and centrifuge. Remove the supernatant first with a micropipette and then with a Hamilton syringe to get the liquid out of the beads.Fractionate the nuclear extract over a 12%to 32%glycerol gradient. Combine and mix glycerol gradient fractions three, four, and five which are near the 10S region. Prepare two 150 microliter aliquots of combined gradient fractions three through five and place them in 50 microliters of anti-gal3 beads.In parallel, prepare two samples each with 150 microliters of fraction one and place them in 50 microliters of anti-gal3 beads. As a control, place 150 microliters of 60%buffer D in 50 microliters of anti-gal3 beads. Mix gently by tapping the tubes, then rotate them head over tail at four degrees Celsius for one hour.Pellet the samples with gentle centrifugation. Remove most of the supernatant with a micropipette. Remove the supernatant using a Hamilton syringe by carefully inserting the needle tip to the bottom of the beads.Use the beads immediately for the splicing reactions. Assemble the splicing reaction as described in the text manuscript and add it to one set of the bead samples. Assemble an identical set of splicing reactions in a total volume of 24 microliters but without U1-depleted nuclear extract and add it to the other set of beads.Prepare a control splicing reaction to be carried out in the absence of any beads in a total volume of 12 microliters. This control reaction contains nuclear extract, 60%buffer D and radioactive splicing substrate. Mix the tubes gently by tapping and rotate them head over tail at 30 degrees Celsius for 90 minutes, then pellet the mixture by gentle centrifugation.Stop the reaction and elute the proteins off the beads by adding 24 microliters of 2X SDS sample buffer to the tubes containing beads and 12 microliters of 2X SDS sample buffer to the control tube containing nuclear extract but no beads. Vortex each tube. Heat the tubes at 100 degrees Celsius for seven minutes.Centrifuge the tubes at 1, 000 times G in a swinging bucket rotor at room temperature for 10 to 15 seconds. Transfer the supernatants to fresh micro tubes. Add 20 milligrams per milliliter proteinase K to digest and solubilize the proteins.Incubate the tubes at 37 degrees Celsius for 40 minutes. After the incubation, gently centrifuge the tubes. Dilute the bead elusions with 39.5 microliters TE and 10 microliters of three molar sodium acetate.Dilute the nuclear extract control with 63.5 microliters TE and 10 microliters of sodium acetate. Extract and analyze the RNA as described in the text manuscript. Nuclear extracts depleted of U1 snRNP and gal3 U1 snRNP complexes from the 10S region of glycerol gradient immunoprecipitated by anti-gal3 were mixed in a splicing reaction.This reaction mixture contained U1 snRNA, as well as the U1 specific protein U1-70K. As expected, the anti-gal3 precipitated gal3. U1 snRNA, U1-70K protein, and gal3 were not found in the pre-immune control precipitation.Compared to a non-depleted nuclear extract carried out as a positive control, the nuclear extract depleted of U1 snRNP did not exhibit splicing activity. Splicing activity in the U1-depleted nuclear extract could be reconstituted by the bead-bound gal3 U1 snRNP complex. Both products of the splicing reaction ligated exons and excised intron lariat as well as in intermediates were found.The components of fractions three through five were critical in restoring splicing to the U1-depleted nuclear extract. When these fractions were replaced by buffer alone, no splicing activity could be observed, indicating that the anti-gal3 beads were not responsible for the restoration of splicing activity in nuclear extract depleted of U1.More persuasively, when fractions three through five were replaced by fraction one in the immunoprecipitation procedure and then added to the U1-depleted nuclear extract, no intermediates or products of the splicing reaction were found. When attempting this protocol, one person should complete the immunoprecipitation while the other person prepares the splicing reaction with as little delay as possible.Proteomic analysis of the polypeptide composition of the galectin-3 U1 SNRP which restores splicing activity to a U1-depleted nuclear extract would be of great interest.

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Biochemistry

2.1K visualizações.  Michigan State University. This report provides the experimental evidence for the documentation that a galectin-3 U1 snRNP complex binds to pre mRNA substrate forms a functional complex and leads to products of the splicing reaction. This protocol utilizes the galectin-3 U1 snRNP immunoselected on beads covalently coupled to anti-gal3 antibodies to initiate the splicing reaction, which can progress to completion. A critical step in the protocol is the careful removal of liquid after pelleting the beads containing the g...