Saccharomyces cerevisiae BY4741 Transcriptome or Gene expression

mRNA Translation plays important roles in eukaryotic gene expression. Upstream Open Reading Frames (uORFs) are regulatory elements located in 5' mRNA transcript leaders that usually reduce translation of downstream main open reading frames (mORFs). While many aspects of uORF function are well studied, the impact of uORF coding sequences has not been systematically evaluated. We investigated this by assaying thousands of variants in the yeast YAP1 uORF with a new massively parallel reporter assay (FACS-uORF). Varying uORF codons resulted in a broad range of functions including both repressors and enhancers. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.