Name: act1-cup1 assays determine the substrate-specific sensitivities of spliceosomal mutants in budding yeast
Uploaded: 2022-06-30T12:30:00
Description: Watch this Scientific Journal Video about ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast at JoVE.com

Thank you to Aaron Hoskins and the Hoskins lab members at the University of Wisconsin-Madison for use of yeast strains and equipment in the generation of figures 3-5. Thank you to Harpreet Kaur and Xingyang Fu for their insightful comments on the manuscript. Thank you to the supportive students, staff, and faculty at Northwest University during the writing, editing, and filming of this paper. Thank you to Isabelle Marasigan for help in filming this method.

1. Yeast strain construction
<ol>
	<li>Generate or obtain an S. cerevisiae strain whose background includes leu2 and cup1&#916;. 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 CUP16,38. Refer to genomic information for the selected yeast...

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...

The spliceosome is a large, biological machine that catalyzes the removal of introns, non-coding regions in precursor messenger RNA (pre-mRNA)1,2.&#160;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.&#160;The change in the mutated component&#39;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&#39;s actin gene, which is highly expressed and has an efficiently spliced intron4,5. Cup1p is a copper chelator that sequesters copper in the cell to prevent interference with regular cellular functions6,7,8. 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&#39;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 mutations3,6,7. 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&#39; splice site (5&#39; SS), branch-site (BS), and 3&#39; splice site (3&#39; 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&#39; SS, specifically position 3 from the consensus adenosine to a cytosine. This reporter will interact strongly with spliceosome mutations that impact 5&#39; SS selection and use. In their initial study, Lesser and Guthrie determined which 5&#39; 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&#39; 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 reporters10,11,12,13,14,15,16,17. Other protein components of the spliceosome have also been investigated using ACT1-CUP1, including Hsh155p, Cwc2p, Cef1p, and Ecm2p18,19,20,21,22,23,24,25. The energetic thresholds for Prp16p and four other ATPases involved in spliceosomal transition have also been studied with this assay9,26,27,28,29,30. 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 splicing3,31,32,33,34,35,36,37.
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 number6,38, preparation of a full knock-out strain can require multiple rounds or extensive screening. As a result, cup1&#916; 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&#39;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&#39;s effect on splicing and which intronic regions are most impacted by the perturbation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL sterile microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>2 mL sterile microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>07-201-332</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>96-well round bottom microplate</td>
			<td>Fisher Scientific</td>
			<td>07-200-760</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>190 proof ethanol</td>
			<td>Fisher Scientific</td>
			<td>22-032-600</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>500 mL Filter System (0.22 &#181;m)</td>
			<td>CellTreat Scientific Products</td>
			<td>229707</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-500</td>
			<td>Any molecular grade agar will work.</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Tuttnauer</td>
			<td>3870EA</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Humboldt</td>
			<td>PN6200.1</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Cell Density Meter</td>
			<td>VWR</td>
			<td>490005-906</td>
			<td>Or other spectral device that can measure absorbance at 595 nm.</td>
		</tr>
		<tr>
			<td>Copper sulfate Pentahydrate</td>
			<td>Fisher Scientific</td>
			<td>LC134051</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Digital imaging system</td>
			<td>Cytiva</td>
			<td>29399481</td>
			<td>ImageQuant 4000 (used for Figure 3),&#160; Amersham ImageQuant 800, or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Dropout mix (-Leu)</td>
			<td>USBiological Life Sciences</td>
			<td>D9525</td>
			<td>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</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Scientific</td>
			<td>AAA1682836</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Gel band quantifying software</td>
			<td>Cytiva</td>
			<td>29-0006-05</td>
			<td>ImageQuant TL v8.1 (used for figure 5A) or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Hand held camera</td>
			<td>Nikon</td>
			<td>D3500</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Near infra-red gel imaging device</td>
			<td>Cytiva</td>
			<td>29238583</td>
			<td>Amersham Typhoon NIR (used for Figure 5a) or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Laboratory grade clamp</td>
			<td>Fisher Scientific</td>
			<td>05-769-7Q</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Laboratory grade stand and clamp</td>
			<td>Fisher Scientific</td>
			<td>12-000-101</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Magnetic stir bars</td>
			<td>Fisher Scientific</td>
			<td>14-513-51</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Pin replicator</td>
			<td>VP Scientific</td>
			<td>VP 407AH</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-micro disposable cuvettes</td>
			<td>VWR</td>
			<td>97000-590</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>JEIO Tech</td>
			<td>IST-3075</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biowave</td>
			<td>80-3000-45</td>
			<td>Or any spectophotometer that can measure the absorbance at 600 nm.</td>
		</tr>
		<tr>
			<td>Square plates</td>
			<td>VWR</td>
			<td>102091-156</td>
			<td>Circular plates may also be used though are more challenging if using a pin replicator.</td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Fisher Scientific</td>
			<td>11-520-16S</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
		<tr>
			<td>Yeast nitrogen base</td>
			<td>USBiological Life Sciences</td>
			<td>Y2025</td>
			<td>Or comparable item from a different manufacturer.</td>
		</tr>
	</tbody>
</table>

act1-cup1 assays determine the substrate-specific sensitivities of spliceosomal mutants in budding yeast

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.

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...

Watch this Scientific Journal Video about ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast at JoVE.com

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

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.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

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.

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