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 strain when designing knock-out primers to flank the CUP1 gene location(s).</li>
	<li>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 research40,41,42.</li>
	<li>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.</li>
	<li>Perform steps 1.2. and 1.3. for each QSP and each ACT1-CUP1 reporter plasmid to be tested, including control strains.</li>
</ol>
2. Copper plate preparation
<ol>
	<li>Select a copper concentration range that suits the reporters to be tested (see Table 1 for frequently used reporters&#39; 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+.</li>
	<li>Make a stock solution of 1 M CuSO4 and sterile filter through a 0.22 &#181;m PES (polyethersulfone) sterile filter.</li>
	<li>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.).
	<ol>
		<li>Calculate the amount of stock for the final desired copper concentration in 40 mL of the plate volume (Supplemental Table 1).</li>
		<li>Add the calculated amount of sterile water and 1 M CuSO4 stock to a sterile, 2 mL tube.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Label a 100 mL bottle per copper concentration to be tested.</li>
		<li>To each bottle, add 790 mg of agar (2% w/v agar) and a stir bar.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Autoclave to sterilize and dissolve the agar using the recommended liquid cycle for the autoclave.</li>
		<li>As promptly as possible, add 4 mL of 20% w/v glucose (sterile filtered) to each bottle.</li>
		<li>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.</li>
		<li>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. 
		&#8203;NOTE: Frequently, the plates are made 1 day or 2 days in advance of the assay and stored at 4 &#176;C until a few hours before use. The plates should be at room temperature (RT) before plating begins (step 3.4.).</li>
	</ol>
	</li>
</ol>
3. ACT1-CUP1 assay
<ol>
	<li>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 &#176;C. Then, restreak a small swatch and allow it to grow for another 2-3 days at 30 &#176;C.</li>
	<li>Grow overnight cultures in 10 mL of media.
	<ol>
		<li>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 &#181;m PES sterile filter.</li>
		<li>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.</li>
		<li>Using a sterile stick or pipet tip, gather a small (~1 mm round) swatch of yeast and inoculate the media.</li>
		<li>Shake all the overnight cultures at 180 rpm and 30 &#176;C. 
		NOTE: If available, rotators can be used instead of a shaker.</li>
	</ol>
	</li>
	<li>Dilute the strains to an OD600 0.5 &#177; 0.05 in 10% glycerol.
	<ol>
		<li>Per strain, add 100 &#181;L of culture to a cuvette containing 900 &#181;L of water.</li>
		<li>Measure the OD600 with a spectrophotometer.</li>
		<li>Calculate the dilution required to be at an OD600 of 0.5 in a final volume of 2 mL.</li>
		<li>Dilute each strain to OD600 0.5 in 10% glycerol (sterile).</li>
		<li>Remeasure the OD600 to confirm the cell density is within the desired range of 0.5 &#177; 0.05.</li>
	</ol>
	</li>
	<li>Plate the strains on the copper plates. 
	&#8203;NOTE: A variety of methods can be used to plate the strains, including hand pipetting 5-10 &#181;L 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.
	<ol>
		<li>Set up a sterile working location and a lit Bunsen burner.</li>
		<li>For a 48-pin replicator, pipet 200 &#181;L 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 &#181;L of 10% glycerol (sterile). 
		NOTE: An example of a plating scheme for nine yeast strains is in Supplemental Table 2.</li>
		<li>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.</li>
		<li>Place four plates near the burner and remove the lids.</li>
		<li>Dip the replicator in the 96-well plate and lift it up in one quick motion.</li>
		<li>Place gently onto the plate and rock lightly back and forth to facilitate a good transfer.</li>
		<li>Lift up in one swift motion and place in the exact same orientation in the 96-well plate.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Let the plates dry completely before placing the lids on, usually 3-5 min.</li>
		<li>Incubate the plates for 3 days at 30 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Data collection and analysis
<ol>
	<li>Remove the plates from the incubator and visually inspect them.</li>
	<li>Record the images of the plates with an available camera or other digital imaging system.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

The author has nothing to disclose.

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><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 &amp;micro;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),&amp;nbsp; 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. ...

act1-cup1-assays-determine-substrate-specific-sensitivities

Research

JoVE Journal

Genetics

2.4K Views.  Northwest University. 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.

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

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Cancer Research

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

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

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.

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

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.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Biochemistry

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

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

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A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

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

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Competitive Genomic Screens of Barcoded Yeast Libraries

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis