| Summary: | Nested protein-coding genes accumulated throughout metazoan evolution, with early analyses of human and <i>Drosophila</i> microarray data indicating that this phenomenon was simply due to the presence of large introns. However, a recent study employing RNA-seq data uncovered evidence of transcriptional interference driving rapid expression divergence between <i>Drosophila</i> nested genes, illustrating that accurate expression estimation of overlapping genes can enhance detection of their relationships. Hence, here I apply an analogous approach to strand-specific RNA-seq data from human and mouse to revisit the role of transcriptional interference in the evolution of mammalian nested genes. A genomic survey reveals that whereas mammalian nested genes indeed accrued over evolutionary time, they are retained at lower frequencies than in <i>Drosophila</i>. Though several properties of mammalian nested genes align with observations in <i>Drosophila</i> and with expectations under transcriptional interference, contrary to both, their expression divergence is not statistically different from that between unnested genes, and also does not increase after nesting. Together, these results support the hypothesis that lower selection efficiencies limit rates of gene expression evolution in mammals, leading to their reliance on immediate eradication of deleterious nested genes to avoid transcriptional interference.
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