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 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. ...

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

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...