Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded. [Display omitted] •TIS-profiling reveals widespread translation of non-canonical ORFs in budding yeast•Production of non-AUG-initiated extended isoforms is prevalent and inefficient•A small subset of possible near-cognate sites is used for translation initiation•eIF5A-based regulation allows conditional unmasking of non-AUG initiation in meiosis Eisenberg et al. identify translation initiation sites genome-wide in budding yeast. They define a class of 149 genes with alternate extended isoforms that initiate at non-AUG start codons. These isoforms are produced in concert with their corresponding canonical isoforms but typically at lower abundance, and are selectively induced during meiosis.