Screening Saccharomyces cerevisiae for small-molecule inhibitors of mRNA 3’ end formation

Abstract

mRNA 3’ end processing is an essential process shared by all eukaryotes and involves cleavage of the mRNAs and the addition of 70-90 adenosine residues. This modification is essential for mRNA biogenesis and gene expression. We developed a powerful screen to identify small molecule that inhibit mRNA 3’ end processing in yeast. Our growth-based assay employs a reporter plasmid with the CYC1 poly(A) site inserted upstream of the CUP1 coding sequence. In yeast carrying the reporter plasmid, defects in mRNA 3’ end processing are known to allow read-through into CUP1, enabling cells to grow in medium containing 0.6mM CuSO4. A high through-put screen of 70,000 compounds led to identification of 28 compounds. Different concentrations of each compound were then tested using the same assay to determine the dose response effect. Of those 28 compounds, 6 were confirmed to have a strong effect. b-galactosidase based assay was used to confirm the compounds that were effective in the growthbased assay and we found one compound (N,N-diethyl-N'-(4-methylbenzothioyl)thiourea) is effective in b-galactosidase assay and also strain defective in cleavage and polyadenylation is more sensitive for this compound more than wild type stain.

Keywords: β-galactosidase assay; high-throughput screening; copper
assay, mRNA 3’ end formation.