ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Summary

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

Abstract

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

Introduction

The spliceosome is a large, biological machine that catalyzes the removal of introns, non-coding regions in precursor messenger RNA (pre-mRNA)^1,2. Characterizing the effect of a single point mutant in 1 of the nearly 100 proteins and 5 non-coding RNAs is often ambiguous when studying the protein or RNA in isolation. The change in the mutated component's function can best be evaluated in vivo in the context of the full, functioning spliceosome.

The copper growth assay described here is a quick gauge of splicing efficiency in Saccharomyces cerevisiae or budding yeast. Developed by C.F. Lesser and C. Guthrie and published in 1993, this assay combines the ease of working with a simple model organism and the straightforward readout of cell viability³. The viability correlates with how well the spliceosomes in these cells can recognize and splice the reporter transcript.

This copper growth assay is more commonly called the ACT1-CUP1 assay. The name ACT1-CUP1 originates from the two genes fused to create a reporter of splicing efficiency. ACT1 is yeast's actin gene, which is highly expressed and has an efficiently spliced intron^4,5. Cup1p is a copper chelator that sequesters copper in the cell to prevent interference with regular cellular functions^6,7,8. The ACT1-CUP1 reporter contains these genes in sequence such that CUP1 is in the proper reading frame only if pre-mRNA splicing of ACT1's intron occurs (Figure 1). The resulting fusion protein contains the first 21 amino acids of actin and the full length Cup1p protein, which increases yeast viability in a copper-rich environment³. Thus, an increase in the amount of splicing of the reporter results in a higher concentration of Cup1p and a higher copper resistance (Figure 1). In comparison to other reporter genes, CUP1 impacts cell viability even at low levels, has a wide sensitivity range, and can be used to directly select for splicing mutations^3,6,7. In addition, CUP1 is non-essential for standard yeast growth, and thus cellular homeostasis is not impacted during the setup for this assay. Complementary to deletion or temperature growth assays, ACT1-CUP1 provides information about the effects on splicing under otherwise optimal yeast growth conditions.

The spliceosome recognizes its substrate through three intronic sequences, namely the 5' splice site (5' SS), branch-site (BS), and 3' splice site (3' SS). Numerous ACT1-CUP1 reporters have been generated containing non-consensus sequences at these sites. A selection of the most common ACT1-CUP1 reporters is shown in Figure 1 and Table 1. As the spliceosome interacts with each splice site uniquely at different points in the splicing cycle, the robustness of the spliceosome can be tested at different steps based on which non-consensus reporter is used. Non-consensus reporters are named for the mutated position within the intron and the base it was mutated to. For example, A3c is a reporter with a mutation at the 5' SS, specifically position 3 from the consensus adenosine to a cytosine. This reporter will interact strongly with spliceosome mutations that impact 5' SS selection and use. In their initial study, Lesser and Guthrie determined which 5' SS mutations inhibited splicing³. Later the same year, non-consensus reporters at all three splice sites were published by Burgess and Guthrie in a suppressor screen of mutations in the ATPase Prp16p⁹. Comparing consensus to non-consensus reporters, the ACT1-CUP1 assay has been an important key to understanding the robustness and selectivity of the yeast spliceosome and to infer the function of other eukaryotes' spliceosomes.

As non-consensus ACT1-CUP1 reporters sensitize the spliceosome to further perturbation, the impact of a single splicing factor mutation can be characterized through the reporters it positively or negatively impacts. This has been applied to splicing research questions in a variety of ways. First, the ACT1-CUP1 assay can and has been used as a genetic screen for mutations in splicing factors. For example, Prp8p, the largest splicing protein, serves as a platform upon which the RNA core of the spliceosome catalyzes the splicing reaction. This was deduced, in part, through how Prp8p mutants improved or reduced the splicing of different ACT1-CUP1 reporters^{10,11,12,13,14,15,16,17}. Other protein components of the spliceosome have also been investigated using ACT1-CUP1, including Hsh155p, Cwc2p, Cef1p, and Ecm2p^{18,19,20,21,22,23,24,25}. The energetic thresholds for Prp16p and four other ATPases involved in spliceosomal transition have also been studied with this assay^{9,26,27,28,29,30}. The small nuclear RNAs (snRNAs) have also been extensively studied utilizing ACT1-CUP1 to identify the pre-mRNA sequences they coordinate and the changes in secondary structure the snRNAs undergo during splicing^{3,31,32,33,34,35,36,37}.

The ACT1-CUP1 assay requires a yeast strain where all copies of the CUP1 gene have been knocked-out. As CUP1 can have a high copy number^6,38, preparation of a full knock-out strain can require multiple rounds or extensive screening. As a result, cup1Δ yeast strains have often been shared between labs, as have the reporters.

If mutation(s) in a splicing factor are being assessed from a plasmid copy, the wild-type gene for this factor should be knocked-out. In addition, the yeast background should allow for the selection of at least two plasmids, one containing an ACT1-CUP1 reporter, historically on a leucine nutrient-selection plasmid, and one containing a mutation or perturbation in the splicing machinery that will be studied (Figure 2). Usually, in a single assay, multiple yeast strains, each carrying the query splicing perturbation (QSP) and a different reporter, will test the query's impact on splicing.

The independent variables in the ACT1-CUP1 assay allow a researcher to assess the severity of a QSP. These independent variables are the concentration of copper and the selection of multiple non-consensus splicing reporters. First, as the yeast strains are grown on plates containing a range of copper concentrations (Figure 2), setting up the assay includes selecting the gradient of concentrations used. Studies can utilize a course copper concentration gradient to get an initial readout of viability and then repeat the assay with a finer gradient to identify subtle viability differences. The second variable is the wide range of ACT1-CUP1 reporters possible to test (Figure 1 and Table 1). If the QSP impacts yeast viability differently in the presence of a non-consensus reporter versus wild-type, a conclusion can be made that the QSP affects a step in splicing or a region of the spliceosome important during the recognition or processing of that region of the intron.

The yeast toolbox is extensive, and the ACT1-CUP1 assay is an integral part of splicing research. The ACT1-CUP1 assay is often performed alongside a more in-depth genetic, structural, and/or biochemical analysis on the impact of a QSP. As these more detailed studies generally have a lengthier procedure and/or higher price tag, a frequent approach is screening for interesting mutants with ACT1-CUP1 first.

Provided here is an ACT1-CUP1 assay protocol, including copper plate preparation. This assay provides researchers with an initial answer to a QSP's effect on splicing and which intronic regions are most impacted by the perturbation.

Protocol

1. Yeast strain construction

1. Generate or obtain an S. cerevisiae strain whose background includes leu2 and cup1Δ. To generate this background, use the well-established yeast method that employs lithium acetate and single-stranded DNA³⁹.
   NOTE: Haploid yeast strains may contain one, two, or more copies of CUP1^6,38. Refer to genomic information for the selected yeast strain when designing knock-out primers to flank the CUP1 gene location(s).
2. Perform a yeast transformation to incorporate the QSP either via genomic incorporation or on a plasmid. Use a well-established protocol such as those described in previous research^40,41,42.
3. Perform a yeast transformation with the resulting strain(s) from step 1.2. to add the desired ACT1-CUP1 reporter plasmid.
   NOTE: Cells must be maintained on leucine drop-out (-Leu) plates and media to ensure retention of the ACT1-CUP1 reporter plasmids following this transformation.
4. Perform steps 1.2. and 1.3. for each QSP and each ACT1-CUP1 reporter plasmid to be tested, including control strains.

2. Copper plate preparation

1. Select a copper concentration range that suits the reporters to be tested (see Table 1 for frequently used reporters' lethality).
   NOTE: An example of a comprehensive copper concentration range is 30 different copper concentrations of 0 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, and 2.5 mM Cu²⁺.
2. Make a stock solution of 1 M CuSO₄ and sterile filter through a 0.22 µm PES (polyethersulfone) sterile filter.
3. Per desired copper plate, prepare a 2 mL dilution of the CuSO₄ stock in sterile water.
   NOTE: As the plate with 0 mM Cu²⁺ will always be analyzed and imaged as a reference, it is recommended to make two 0 mM Cu²⁺ plates, one at the beginning and one at the end of the plating step (step 3.4.).
   1. Calculate the amount of stock for the final desired copper concentration in 40 mL of the plate volume (Supplemental Table 1).
   2. Add the calculated amount of sterile water and 1 M CuSO₄ stock to a sterile, 2 mL tube.
4. Pour plates for the ACT1-CUP1 assay.
   NOTE: An alternative to the protocol below is to initially combine the media and agar in a large container and aliquot after autoclaving into smaller containers to achieve different copper concentrations for each plate. Whichever method is taken, it is important to ensure that the media concentration is consistent between all of the plates despite each having a different copper concentration.
   1. Label each empty plate to be poured with the final copper concentration it will contain. Prepare at least one square plate per copper concentration to be tested.
   2. Label a 100 mL bottle per copper concentration to be tested.
   3. To each bottle, add 790 mg of agar (2% w/v agar) and a stir bar.
   4. In a large beaker, combine the -Leu growth media for all the copper plates to be poured. Per plate to be made, dissolve 265 mg of yeast nitrogen base (YNB) and 64 mg of drop-out mix minus leucine (and any other nutrients that may be required to maintain the QSP plasmid in the cells) in 34 mL of deionized water.
   5. Add 34 mL of the -Leu growth media solution to each prepared 100 mL bottle and cap with aluminum foil. Label the foil with the intended copper concentration.
   6. Autoclave to sterilize and dissolve the agar using the recommended liquid cycle for the autoclave.
   7. As promptly as possible, add 4 mL of 20% w/v glucose (sterile filtered) to each bottle.
   8. Match the labels and add the 2 mL dilutions of CuSO₄ to its intended bottle.
      NOTE: As tens of copper plates can be made at the same time, each with a different concentration, labeling all bottles, tubes, and plates clearly with the intended copper concentration will prevent confusion during plate pouring.
   9. Use a stir plate to mix for ~30 s and pour or pipet 35 mL into the labeled plate, avoiding bubbles. Allow to cool before storing or use.
      ​NOTE: Frequently, the plates are made 1 day or 2 days in advance of the assay and stored at 4 °C until a few hours before use. The plates should be at room temperature (RT) before plating begins (step 3.4.).

3. ACT1-CUP1 assay

1. Streak out the desired strains on -Leu plates.
   NOTE: If working from cryo stocks, care should be taken to ensure the cells are sufficiently revived from storage before plating. A recommended procedure for this is to streak from the cryogenic stock and allow it to grow for 3-5 days at 30 °C. Then, restreak a small swatch and allow it to grow for another 2-3 days at 30 °C.
2. Grow overnight cultures in 10 mL of media.
   1. Prepare -Leu growth media using the same ratios as described in step 2.4.2. Per 10 mL of media, add 66 mg of yeast nitrogen base (YNB) and 16 mg of drop-out mix minus leucine to 9 mL of deionized water. Pass through a 0.22 µm PES sterile filter.
   2. Per yeast strain, add 9 mL of -Leu growth media and 1 mL of 20% w/v glucose (sterile filtered) to a sterile 50 mL conical tube.
   3. Using a sterile stick or pipet tip, gather a small (~1 mm round) swatch of yeast and inoculate the media.
   4. Shake all the overnight cultures at 180 rpm and 30 °C.
      NOTE: If available, rotators can be used instead of a shaker.
3. Dilute the strains to an OD₆₀₀ 0.5 ± 0.05 in 10% glycerol.
   1. Per strain, add 100 µL of culture to a cuvette containing 900 µL of water.
   2. Measure the OD₆₀₀ with a spectrophotometer.
   3. Calculate the dilution required to be at an OD₆₀₀ of 0.5 in a final volume of 2 mL.
   4. Dilute each strain to OD₆₀₀ 0.5 in 10% glycerol (sterile).
   5. Remeasure the OD₆₀₀ to confirm the cell density is within the desired range of 0.5 ± 0.05.
4. Plate the strains on the copper plates.
   ​NOTE: A variety of methods can be used to plate the strains, including hand pipetting 5-10 µL volumes, using a repeat or multi-channel pipettor, or stamping with a pin replicator. This last method is described below, though most steps will be similar regardless of the method.
   1. Set up a sterile working location and a lit Bunsen burner.
   2. For a 48-pin replicator, pipet 200 µL of each diluted strain into a separate well of a 96-well plate. Fill the empty spaces in the 6 x 8 grid with 200 µL of 10% glycerol (sterile).
      NOTE: An example of a plating scheme for nine yeast strains is in Supplemental Table 2.
   3. Dip the replicator in a shallow dish of 95% (v/v) ethanol and flame to sterilize. Let it cool for at least 2 min after the flame extinguishes to avoid heat shocking the cells.
   4. Place four plates near the burner and remove the lids.
   5. Dip the replicator in the 96-well plate and lift it up in one quick motion.
   6. Place gently onto the plate and rock lightly back and forth to facilitate a good transfer.
   7. Lift up in one swift motion and place in the exact same orientation in the 96-well plate.
   8. Repeat for up to three other plates. Repeat the process of dipping the replicator in ethanol, flaming to sterilize, and waiting to cool every four plates.
   9. After a plate has been plated, move with a smooth motion to the side but still within the sterilization umbrella of the flame.
      NOTE: It is recommended to do the yeast dilutions and plating near a flame. The plates can easily become contaminated while drying.
   10. Let the plates dry completely before placing the lids on, usually 3-5 min.
   11. Incubate the plates for 3 days at 30 °C.

4. Data collection and analysis

1. Remove the plates from the incubator and visually inspect them.
2. Record the images of the plates with an available camera or other digital imaging system.
3. Record (or score) for each strain the last copper concentration visible growth is observed.
   NOTE: The cells are able to splice and remain viable up until that concentration. For consistency, always use the same method, either by eye or from the plate images, to score the last viable copper concentration. Very small colonies are sometimes visible by the eye but not on the image. The difference between direct visual inspection or recording from images is small, usually a step in the gradient. As images of the colonies are often used in publications, scoring by the images is recommended.
4. Combine data from multiple ACT1-CUP1 assays for the same strain to draw conclusions about how the QSP affects splicing.
   NOTE: Publication figures customarily show images of the yeast colonies at 0 mM Cu²⁺ concentration, the last viable copper concentration, and the subsequent concentration where the colony has died. Data can also be displayed as a bar graph with error bars for the standard deviation between replicates. Data do not need to be normalized but can be by setting the viability of the WT splicing factor control to 1 and comparing the effect of the mutation(s) introduced.

Results

Growth assays, like ACT1-CUP1, require the visual, comparative assessment of multiple colonies. Here, each strain was grown to saturation overnight, diluted to an OD₆₀₀ of 0.5, and plated on 20 plates containing a range of copper concentrations from 0 mM to 1.1 mM CuSO₄ (Figure 3). This range is smaller than that listed in the protocol as it allowed for the full assessment of the impact of the QSPs and ACT1-CUP1 reporters used and described below. The plates were imaged...

Discussion

ACT1-CUP1 is a growth assay, and care must be taken to ensure that observed growth differences can only be attributed to splicing defects. All strains should be handled in a similar fashion prior to plating, including having a similar length and type of growth and storage conditions. If using temperature-sensitive strains, ACT1-CUP1 assays should only be performed under conditions where those strains grow comparably to wild type. Relatedly, for the QSP component, it is advised to have identical yeast backgrounds and expr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL sterile microcentrifuge tubes	Fisher Scientific	05-408-129	Or comparable item from a different manufacturer.
2 mL sterile microcentrifuge tubes	Fisher Scientific	05-408-138	Or comparable item from a different manufacturer.
50 mL sterile centrifuge tubes	Fisher Scientific	07-201-332	Or comparable item from a different manufacturer.
96-well round bottom microplate	Fisher Scientific	07-200-760	Or comparable item from a different manufacturer.
190 proof ethanol	Fisher Scientific	22-032-600	Or comparable item from a different manufacturer.
500 mL Filter System (0.22 µm)	CellTreat Scientific Products	229707	Or comparable item from a different manufacturer.
Agar	Fisher Scientific	BP1423-500	Any molecular grade agar will work.
Autoclave	Tuttnauer	3870EA	Or comparable item from a different manufacturer.
Bunsen burner	Humboldt	PN6200.1	Or comparable item from a different manufacturer.
Cell Density Meter	VWR	490005-906	Or other spectral device that can measure absorbance at 595 nm.
Copper sulfate Pentahydrate	Fisher Scientific	LC134051	Or comparable item from a different manufacturer.
Digital imaging system	Cytiva	29399481	ImageQuant 4000 (used for Figure 3),  Amersham ImageQuant 800, or comparable item from a different manufacturer.
Dropout mix (-Leu)	USBiological Life Sciences	D9525	Use the appropriate drop out mix for your experiment. It is possible you will be using a yeast nutrient marker for your query perturbation also. In that case, the drop out mix should be for that marker and Leu
D-Glucose	Fisher Scientific	AAA1682836	Or comparable item from a different manufacturer.
Gel band quantifying software	Cytiva	29-0006-05	ImageQuant TL v8.1 (used for figure 5A) or comparable item from a different manufacturer.
Hand held camera	Nikon	D3500	Or comparable item from a different manufacturer.
Near infra-red gel imaging device	Cytiva	29238583	Amersham Typhoon NIR (used for Figure 5a) or comparable item from a different manufacturer.
Laboratory grade clamp	Fisher Scientific	05-769-7Q	Or comparable item from a different manufacturer.
Laboratory grade stand and clamp	Fisher Scientific	12-000-101	Or comparable item from a different manufacturer.
Magnetic stir bars	Fisher Scientific	14-513-51	Or comparable item from a different manufacturer.
Pin replicator	VP Scientific	VP 407AH
Semi-micro disposable cuvettes	VWR	97000-590	Or comparable item from a different manufacturer.
Shaker	JEIO Tech	IST-3075	Or comparable item from a different manufacturer.
Spectrophotometer	Biowave	80-3000-45	Or any spectophotometer that can measure the absorbance at 600 nm.
Square plates	VWR	102091-156	Circular plates may also be used though are more challenging if using a pin replicator.
Stir plate	Fisher Scientific	11-520-16S	Or comparable item from a different manufacturer.
Yeast nitrogen base	USBiological Life Sciences	Y2025	Or comparable item from a different manufacturer.