The ACT1-CUP1 Assay can identify if a mutation affects pre-messenger RNA splicing, and give an indication of which splicing step is most impacted. The ACT1-CUP1 Assay links splicing factor mutation to its impact on the complex process of splicing. It utilizes the simple readout of growth phenotype and has a wide sensitivity range.
Efficient use of time is important when carrying out an ACT1-CUP1 Assay. Start with an assay of five to 10 strains to minimize stress-induced growth aberrations. Demonstrating the process will be myself and Isabelle Marasigan, an undergraduate researcher from my laboratory.
To begin, add 790 milligrams of agar and a stir bar to each bottle. In a large beaker, prepare the leucine dropout growth media for all the copper plates to be poured. This includes 265 milligrams of yeast nitrogen base, 64 milligrams of dropout mix, minus leucine, and 34 milliliters of deionized water for each plate to be poured.
Stir to dissolve Then, add 34 milliliters of the leucine dropout growth media solution to each prepared 100-milliliter bottle, and cap with aluminum foil. Next, autoclave to sterilize and dissolve the agar, using the recommended liquid cycle for the autoclave. As promptly as possible after autoclaving, add four milliliters of 20%glucose to each bottle.
Then, match the tube label to the bottle label to add the two milliliters dilutions of copper sulfate each to its intended bottle. Using a stir plate, mix for approximately 30 seconds and pour or pipette 35 milliliters into the labeled plate, avoiding bubbles. Per yeast strain, add nine milliliters of leucine dropout growth media, and one milliliter of 20%glucose to a sterile, 50-milliliter conical tube.
Using a sterile stick, or pipette tip, gather a small swatch of yeast and inoculate the media. Then, shake all the overnight cultures at 180 RPM and 30 degrees Celsius. Next, add 100 microliters of culture to a cuvette, containing 900 microliters of water per strain.
Afterward, measure the optical density with a spectrophotometer and calculate the dilution required to be at an optical density of 0.5 in a final volume of two milliliters. Then, dilute each strain to optical density 0.5 in 10%glycerol and remeasure the optical density to confirm the cell density is within the desired range. After setting up a sterile working location, light a Bunsen burner.
For a 48-pin Replicator, pipette 200 microliters of each diluted strain into a separate well of a 96-well plate, and fill the empty spaces in the six-by-eight grid with 200 microliters of 10%glycerol. Then, dip the replicator in a shallow dish of 95%ethanol and flame to sterilize. Let it cool for at least two minutes after the flame extinguishes to avoid heat shocking the cells.
Place four plates near the burner and remove the lids. Dip the replicator in the 96-well plate and lift it up in one quick motion. Then, place gently onto the plate and rock lightly back and forth to facilitate a good transfer.
Lift up in one swift motion and place in the exact same orientation in the 96-well plate. After a plate has been plated, move with a smooth motion to the side, but still within the sterilization umbrella of the flame. Let the plates dry completely before placing the lids on, usually three to five minutes.
Then, incubate the plates for three days at 30 degrees Celsius. After removing the plates from the incubator, visually inspect them. The growth assay demonstrated that as the copper concentration increases, strains with lower splicing, show decreased confluency, to the point where the copper concentration becomes lethal.
The yeast background includes a disrupted ADE2 gene, resulting in the colonies being varying shades of red. And the mature colonies were deep red in color. While plating, if the replicator is lifted up while moving left or right, brought at an angle, or if shaken above the plate, it is possible to create colonies that are oval shaped, or micro colonies from small droplets of cell solution.
Yeast with the A3c reporter survived to 0.15 millimolar copper while the wild-type reporter cells maintained viability to the end of the range of copper concentrations tested. The assay results showed that the different bases at U6 snRNA position 57 have unique impacts on the spliceosome in combination with a 5 prime, or branch site mutation. While U57c is an additive mutation, decreasing copper tolerance, in combination with A3c and branch site G reporters.
In contrast, U57a increases copper tolerance because it promotes progression to the second step. After splicing perturbation is identified through ACT1-CUP1, research can move to more quantitative methods, like primer extension and the identification of disrupted molecular interaction through EMSAs and enzymatic probing. ACT1-CUP1 has been a foundational assay, building our understanding of the robustness and selectivity of the yeast spliceosome.
And it continues to expand our comprehension of spliceosomal function.
