Genome-wide comparative analysis reveals distinct evolutionary patterns of overlapping genes in prokaryotes and eukaryotes.

Overlapping genes are widely distributed across the genomes of nearly all living organisms. Although thousands of overlapping genes have been identified and studied in several species, a comprehensive annotation of these genes is lacking, and the forces shaping their evolution remain poorly understood. Here, we present a comparative analysis of overlapping genes in 253 prokaryotic genomes, 115 plant genomes, and 111 animal genomes. We find that co-oriented overlapping genes predominate in prokaryotes, whereas nested overlapping genes are the most common type in eukaryotes. In prokaryotes, overlapping genes are significantly shorter than non-overlapping genes, and 69% of co-oriented overlaps occur in translational phase 2. The number of overlapping genes is strongly correlated with genome size in prokaryotes but not in eukaryotes. We estimated the rates at which overlapping genes are gained and lost over evolutionary time and found that loss of overlapping orthologs is the highest rate in eukaryotes, whereas the highest rate in prokaryotes is the gain of overlapping genes from adjacent genes. Expression analyses revealed that, compared with neighboring genes, nested genes exhibit lower expression levels, altered expression correlations, and higher tissue-specific expression patterns in selected eukaryotic species. Based on these patterns, we propose distinct models to explain the evolutionary pressures acting on overlapping genes in prokaryotes and eukaryotes. Together, our findings provide new insights into the evolution of overlapping genes.