Exploring the eukaryotic gene expression machinery using a single-cell yeast gene expression reporter

It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter employing multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 non-essential yeast genes not only revealed striking phenotypic overlaps between similar functions, but also provided us a dataset in which to compare additional genetic or chemical perturbations. Utilizing a binning and clustering algorithm we developed we are able to compare reporter expression data from thousands of mutants in a semi-automated fashion, essentially grouping mutants or conditions based on the levels of reporter signal. I further utilized our reporter to screen a vast library of in vivo produced cyclic peptides using fluorescence-activated cell sorting (FACS), identifying a cyclic peptide that resulted in mild gene-specific pre-mRNA splicing inhibition. Additionally, I adapted our reporter assay to perform a high-throughput small molecule screen to identify inhibitors or modulators of specific gene expression processes. Our efforts led to the identification of a small molecule that inhibits pre-mRNA splicing in a dose-dependent manner. Moreover, I utilized our reporter system to quickly identify loss-of-function mutants in the poorly characterized gene SWM2. The three mutants of interest have reduced interaction with Tgs1p, the conserved trimethylguanosine synthase, which we believe leads to decreased hypermethylation of the 5’ caps of spliceosomal snRNAs. Altogether this work describes the development, validation and utility of the versatile gene expression reporter system we developed, providing our lab and others a valuable tool to interrogate a wide-range of gene expression processes in yeast.