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.
