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 method⁴

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....

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