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

Summary

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.

Abstract

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.

Introduction

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-specif....

Protocol

1. Notes on general procedures

1. 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).
2. Bake all glassware (beakers, flasks, bottles, pipettes, etc.) for a minimum of 4 h at 177 °C. Wrap other utensils (spatulas, stir-bars, etc.) in aluminum foil before baking under the same conditions.
3. Prepare a 0.1% (vol/vol) solution of di....

Results

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.......

Discussion

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.......

Materials

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