ABSTRACT: Bicistronic transcripts (operon-like transcripts) have occasionally been reported in eukaryotes, including unicellular yeasts, plants, and humans, despite the fact that they lack trans-splice mechanisms. However, the characteristics of eukaryotic bicistronic transcripts are poorly understood, except for those in nematodes. Here, we describe the genomic, transcriptomic, and ribosome profiling features of bicistronic transcripts in unicellular yeasts. By comparing the expression level of bicistronic transcripts with their monocistronic equivalents, we identify two main categories of bicistronic transcripts: highly and lowly expressed. These two categories exhibit quite different features. First, highly expressed bicistronic transcripts have higher conservation within and between strains and shorter intergenic spacers with higher GC content and less stable secondary structure. Second, genes in highly expressed bicistronic transcripts have lower translation efficiency, with the second gene showing statistically significant lower translation efficiency than the first. Finally, the genes found in these highly expressed bicistronic transcripts tend to be younger, with more recent origins. Together, these results suggest that bicistronic transcripts in yeast are heterogeneous. We further propose that at least some highly expressed bicistronic transcripts appear to play a role in modulating monocistronic translation.IMPORTANCE Operons, where a single mRNA transcript encodes multiple adjacent proteins, are a widespread feature of bacteria and archaea. In contrast, the genes of eukaryotes are generally considered monocistronic. However, a number of studies have revealed the presence of bicistronic transcripts in eukaryotes, including humans. The basic features of these transcripts are largely unknown in eukaryotes, especially in organisms lacking trans-splice mechanisms. Our analyses characterize bicistronic transcripts in one such eukaryotic group, yeasts. We show that highly expressed bicistronic transcripts have unusual features compared to lowly expressed bicistronic transcripts, with several features influencing translational modulation.
