Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Summary

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.

Abstract

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.

Introduction

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 others^1,2,3; 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 method^4,5 to stabilize the interaction of the yeast splicing factor Cwc24 with the B^act 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’ 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 crosslinks^4,5, 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 complexes^7,8. 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.

Protocol

1. Yeast total extract preparation

1. 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 µg/mL), leucine (30 µg/mL), tryptophan (30 µg/mL), up to OD₆₀₀ = 1.0.
2. Collect the cells from the 1 L culture by centrifuging in three 500 mL centrifuge bottles at 17,000 x g for 10 min at 4 °C and wash twice with 10 mL cold sterile water.
3. Resuspend the collected yeast cells in 1/10 of the cell volume of cold buffer A (10 mmol L^-1 HEPES pH = 7.9, 1.5 mmol L^-1 MgCl₂, 50 mmol L^-1 KCl, 5% v/v glycerol, 0.5 mmol L^-1 DTT, EDTA-free Protease Inhibitor Cocktail).
4. Freeze small drops of cell suspension in liquid nitrogen. The small drops can be obtained by pipetting the resuspended cell solution and dropping 50 µL directly into liquid nitrogen.
   CAUTION: Liquid nitrogen can cause injuries in contact with skin or eyes. Handle using appropriated safety equipment.
   NOTE: The protocol can be paused here. The frozen drops of cell suspension can be stored at -80 °C.
5. Prepare yeast cells lysates by grinding in a Ball Mill device through six cycles at 20 Hz/s for 3 min.
   NOTE: After each cycle, immerse the container harboring the frozen cells in liquid nitrogen to avoid melting. The protocol can be paused here. The frozen extracts can be stored at -80 °C. Yeast splicing extracts can also be prepared using homogenizers to lysate spheroplasts¹⁰, or using mortar and pestle^11,12.
6. Melt the extract by placing the tubes containing them in water at room temperature, shaking occasionally.
7. Centrifuge extracts at 45,000 x g for 1 h at 4 °C.
8. Quantify the protein content of the cleared supernatant by the BCA method¹³.
9. Prepare aliquots of the extracts, fast freeze them in liquid nitrogen and store at -80 °C.
   NOTE: The protocol can be paused here.

2. Glycerol gradient preparation

1. Prepare two glycerol solutions in Buffer A, one containing 10% v/v and the other containing 30% v/v glycerol.
2. Add the crosslinking agent glutaraldehyde to 0.1% v/v in the 30% v/v glycerol solution and mix to homogenize.
   CAUTION: Handle glutaraldehyde inside the fume hood using appropriate safety equipment.
3. Add 6 mL of the cold 10% v/v glycerol solution at the bottom of the 12 mL centrifuge tube (14 x 89 mm).
4. Add 6 mL of the cold 30% v/v glycerol solution supplemented with glutaraldehyde with a syringe attached to a long needle provided with the Gradient Master device at the bottom of the tube, just beneath the 10% v/v glycerol solution.
   NOTE: Alternatively, the 10% v/v glycerol solution can be carefully pipetted on the top of the 30% v/v glycerol/glutaraldehyde solution.
5. Use the Gradient Master device to generate a continuous density gradient.
   NOTE: In the Gradient Master device, the tubes are placed into an appropriate rack and rotated briefly, following the manufacturer’s recommendations for determining the parameters (time/angle/speed). In this work we used: 2:25 min/81.5°/11 rpm.
6. Carefully add 200 μL of a 7% v/v glycerol/buffer A cushion on the top just before the addition of the cell extracts.
   NOTE: The glycerol concentration of the cushion must be lower than the less concentrated glycerol solution used to create the linear gradient.

3. Extracts centrifugation

1. Load approximately 2 mg of total protein on the top of each 12 mL linear glycerol gradient 10%–30% glutaraldehyde with cushion.
2. Place the tubes in a pre-cooled swing-bucket rotor.
   NOTE: Handle the tubes gently to avoid mixing before ultracentrifugation.
3. Centrifuge at 194,000 x g for 16 h at 4 °C.
4. Aliquot each 12 mL tube in twenty-four 500 μL fractions using an adapted EconoSystem or by carefully pipetting.
   NOTE: An adapted EconoSystem consists of a peristaltic pump, the UV detector, and the fraction collector connected to the tube-perforating device. The sedimentation profile can be monitored by the measurement of the absorbance at 280 nm.
5. Use a 40% v/v glycerol solution through the peristaltic pump to push the glycerol 10%–30% gradient from the tube to the fraction collector.
   NOTE: The protocol can be paused here. Store the fractions at -80 °C until use.
6. Load 50 μL of each fraction directly on nitrocellulose membranes on a dot blot or slot blot device. Detect protein by immunoblot using antibody against CBP (1:6,000, to detect the CBP portion of the TAP tag) and anti-rabbit IgG conjugated with Horseradish Peroxidase (1:15,000) as the secondary antibody.

Results

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 μL 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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Anti-Calmodulin Binding Protein Epitope	Millipore	07-482
ECL anti-Rabbit IgG	GE Healthcare	NA934
EconoSystem	Bio-Rad	1-800-424-6723	Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector
EDTA-free Protease Inhibitor Cocktail	Roche	11873580001
Fraction Recovery System	Beckman Coulter	270-331580	Tube-perforating device that was connected to the parts of the EconoSystem
Gradient Master Model 107ip	Biocomp	107-201M
Mixer Mill MM 200	Retsch	207460001	Ball Mill device
Rotor F12-6x500Lex	Thermo Scientific	096-062375
Sorvall RC 6 Plus Centrifuge	Thermo Scientific	36-101-0816
Swinging Bucket Rotor P40ST	Hitachi
Ultracentrifuge CP 80 NX	Hitachi	901069
Ultra-Clear Centrifuge Tubes (14 x 89 mm)	Beckman Coulter	344059