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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 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.
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.
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 (αU1 beads). The unbound fraction is depleted of U1 snRNP (U1ΔNE). (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 (αGal3 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 U1ΔNE from Part (A) in a splicing assay using 32P-labeled MINX pre-mRNA substrate and the intermediates and products of the splicing reaction are analyzed by gel electrophoresis and autoradiography. Please click here to view a larger version of this figure.
1. Notes on general procedures
Name of buffer | Composition |
Borate buffer | 0.2 M sodium borate, pH 9 |
Buffer C | 20 mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM dithiothreitol (DTT) |
Buffer D | 10 mM HEPES, pH 7.9, 20% (vol/vol) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT |
60%D | 60% Buffer D and 40% H2O |
Ethanolamine | 0.2 M ethanolamine, pH 8 |
HEPES binding buffer | 20 mM HEPES, pH 7.9 |
HEPES wash buffer | 20 mM HEPES, pH 7.9, 0.5 M NaCl |
RNA loading buffer | 90% formamide, 20 mM EDTA, pH 8, 0.05% (w/v) bromophenol blue |
SDS-PAGE buffer | 25 mM Tris, 169 mM glycine, 0.1% sodium dodecyl sulfate (SDS), pH 8.8 |
SDS sample buffer | 62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.1% (w/v) bromophenol blue |
TBE buffer for RNA gels | 89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3 |
TE buffer | 10 mM Tris, pH 8, 1 mM EDTA |
Transfer buffer | 25 mM Tris, 1.92 M glycine, 20% methanol, pH 8.3 |
T-TBS buffer | 10 mM Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.5 |
TX wash buffer | 0.05% Triton X-100 (TX) in 60%D |
Table 1: Name and Composition of Buffers
2. Preparation of NE depleted of U1 snRNP (U1 ΔNE)
3. Immunoprecipitation of 10S fractions of glycerol gradients by anti-Gal3
4. Assembly of splicing reaction and analysis of products
NE depleted of U1 snRNP (U1ΔNE 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. 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...
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...
The authors have nothing to disclose.
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).
Name | Company | Catalog Number | Comments |
anti-U1 snRNP | The Binding Site | Hu ENA-RNP #33471 | human autoimmune serum specific for U1 snRNP |
bottle top vacuum filter | Fisher Scientific | Corning 431153 (0.22 μm; PES 150 ml) | for filtering solutions containing Tris |
centrifuge | International Equipment Company | IEC Model PR-6 | for pelletting Sepharose beads in immunoprecipitation |
diethylpyrocarbonate (DEPC) | Sigma-Aldrich | 159220-5G | for treatment of water used in preparation of all solutions |
dimethylpimelimidate (DMP) | Sigma-Aldrich | 80490-5G | for cross-linking antibody to Sepharose beads |
electrophoresis cell | BioRad Laboratories, Inc | Mini-Protean II | for SDS-PAGE separation of proteins |
ethanolamine | Sigma-Aldrich | 411000-100ml | for blocking after the cross-linking reaction |
gel electrophoresis system | Hoefer, Inc | HSI SE 500 Series | for separating snRNAs by gel electrophoresis |
gel slab dryer | BioRad | Model 224 | for drying gel slabs for autoradiography |
Hybond ECL membrane | GE Healthcare | RPN3032D (0.2 μm; 30 cm x 3 m) | for immunoblotting of proteins on membrane |
microdialyzer (12 x 100 μl sample capacity) | Pierce | Microdialyzer System 100 | for exchanging the buffer of nuclear extract |
microdialyzer membranes (8K cutoff) | Pierce | 66310 | for exchanging the buffer of nuclear extract |
non-fat dry milk | Spartan Stores | Spartan Instant Non-fat Dry Milk | |
Protein A Sepharose CL-4B | Millipore-Sigma | GE 17-0780-01 | for coupling antibody to beads |
Proteinase K | Millipore-Sigma | P2308-5mg | for stopping the splicing reaction to isolate the RNAs |
RNasin | Promega | N2111 | for inhibiting ribonuclease activity |
rocker/rotator | Lab Industries, Inc | Labquake Shaker 400-110 | for mixing protein solutions in coupling reactions and in immunoprecipitation |
Safety-Solve | Research Products International Corp. | No. 111177 | scintillation counting cocktail for determination of radioactivity in splicing substrate |
scintillation counter | Beckman Instruments | LS6000SC | scintillation counter for determination of radioactivity |
speed vaccum concentrator | Savant | SVC 100H | for drying ethanol-precipitated RNA pellets |
Transphor electrophoresis unit | Hoefer, Inc | Hoefer TE Series Transphor | for protein transfer from SDS-PAGE to blotting membrane |
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