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

Summary

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

Abstract

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

Introduction

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

The copper growth assay described here is a quick gauge of splicing efficiency in Saccharomyces cerevisiae or....

Protocol

1. Yeast strain construction

1. Generate or obtain an S. cerevisiae strain whose background includes leu2 and cup1Δ. To generate this background, use the well-established yeast method that employs lithium acetate and single-stranded DNA³⁹.
   NOTE: Haploid yeast strains may contain one, two, or more copies of CUP1^6,38. Refer to genomic information for the selected yeast strain when designing knock-out primers to flank the CUP1 gene location(s).
2. Perform a yeast transformation to incorporate the QSP ....

Results

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

Discussion

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

Materials

List of materials used in this article

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