Determinants of genome-wide distribution and evolution of uORFs in eukaryotes

Upstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze ~17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (N(e)), has shaped the contents of uORFs in eukaryotes. Besides, gene expression level is important in influencing uORF occurrences across genes in a species. Our analyses suggest that most uORFs might play regulatory roles rather than encode functional peptides. We also show that the Kozak sequence context of uORFs has evolved across eukaryotic clades, and that noncanonical uORFs tend to have weaker suppressive effects than canonical uORFs in translation regulation. This study provides insights into the driving forces underlying uORF evolution in eukaryotes.