Detecting Functional Divergence after Gene Duplication through Evolutionary Changes in Posttranslational Regulatory Sequences

Gene duplication is an important evolutionary mechanism that can result in functional divergence in paralogs due to neo-functionalization or sub-functionalization. Consistent with functional divergence after gene duplication, recent studies have shown accelerated evolution in retained paralogs. However, little is known in general about the impact of this accelerated evolution on the molecular functions of retained paralogs. For example, do new functions typically involve changes in enzymatic activities, or changes in protein regulation? Here we study the evolution of posttranslational regulation by examining the evolution of important regulatory sequences (short linear motifs) in retained duplicates created by the whole-genome duplication in budding yeast. To do so, we identified short linear motifs whose evolutionary constraint has relaxed after gene duplication with a likelihood-ratio test that can account for heterogeneity in the evolutionary process by using a non-central chi-squared null distribution. We find that short linear motifs are more likely to show changes in evolutionary constraints in retained duplicates compared to single-copy genes. We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation. Finally, we experimentally confirm our prediction that for the Ace2/Swi5 paralogs, Cbk1 regulated localization was lost along the lineage leading to SWI5 after gene duplication. Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.Author Summary: How a protein is controlled is intimately linked to its function. Therefore, evolution can drive the functional divergence of proteins by tweaking their regulation, even if enzymatic capacities are preserved. Changes in posttranslational regulation (protein phosphorylation, degradation, subcellular localization, etc.) could therefore represent key mechanisms in functional divergence and lead to different phenotypic outcomes. Since disordered protein regions contain sites of protein modification and interaction (known as short linear motifs) and evolve rapidly relative to domains encoding enzymatic functions, these regions are good candidates to harbour sequence changes that underlie changes in function. In this study, we develop a statistical framework to identify changes in rate of evolution specific to protein regulatory sequences and identify hundreds of short linear motifs in disordered regions that are likely to have diverged after the whole-genome duplication in budding yeast. We show that these divergent motifs are much more frequent in paralogs than in single-copy proteins, and that they are more frequent in duplicate pairs that have functionally diverged. Our analysis suggests that changes in short linear motifs in disordered protein regions could be important molecular mechanisms of functional divergence after gene duplication.