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Detecting functional divergence after gene duplication through evolutionary changes in posttranslational regulatory sequences.

Nguyen Ba AN, Strome B, Hua JJ, Desmond J, Gagnon-Arsenault I, Weiss EL, Landry CR, Moses AM - PLoS Comput. Biol. (2014)

Bottom Line: 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.Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Systems Biology, University of Toronto, Toronto, Canada; Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Canada.

ABSTRACT
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 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.

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Examples of known regulatory motifs with changes in constraints.Alignment of the short linear motifs with known function (indicated with arrows) and significant changes in constraints (red boxes) after gene duplication from representative species. A) The Rck2 protein is known to bind and be phosphorylated by Hog1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Rck2 protein. The two identified motifs occur at aa519-538 and aa577-591 for Rck2, and changed constraints within the aligned region aa439-456 and aa492-506 in Rck1. These overlap with the known phosphorylation site in Rck2 (aa520) and the MAP kinase binding site (aa492-506) in Rck2. Both Rck2 and Rck1 retain kinase function. B) The Fkh2 protein is known to be phosphorylated by Cdk1 at two phosphorylation sites on a region shown to have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Fkh2 protein. The identified motif occurs at region aa692-702 in Fkh2 and has changed constraint in the aligned region aa459-469 in Fkh1. One of the known phosphorylation site in Fkh2 occurs within this region at aa697. Fkh2 and Fkh1 retain their forkhead-associated domain (FHA) and DNA binding domain (DBD). C) The Ace2 protein is known to bind and be phosphorylated by Cbk1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Ace2 protein. The two identified motifs occur at aa121-134 and aa283-287 in Ace2, and changed constraints within the aligned region aa115-128 and aa247-248 (it is a gap) in Swi5. These overlap with the known phosphorylation site in Ace2 (aa122) and the Cbk1 binding site (aa283-286) in Ace2. Both Ace2 and Swi5 retain their DNA binding domain (DBD). Stars represent significant changes in constraints along the lineage. Red double arrow illustrates the duplication event. aa: amino acid position. Scer: S. cerevisiae, Ncas: N. castellii, Zrou: Z. rouxii, Calb: C. albicans.
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pcbi-1003977-g004: Examples of known regulatory motifs with changes in constraints.Alignment of the short linear motifs with known function (indicated with arrows) and significant changes in constraints (red boxes) after gene duplication from representative species. A) The Rck2 protein is known to bind and be phosphorylated by Hog1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Rck2 protein. The two identified motifs occur at aa519-538 and aa577-591 for Rck2, and changed constraints within the aligned region aa439-456 and aa492-506 in Rck1. These overlap with the known phosphorylation site in Rck2 (aa520) and the MAP kinase binding site (aa492-506) in Rck2. Both Rck2 and Rck1 retain kinase function. B) The Fkh2 protein is known to be phosphorylated by Cdk1 at two phosphorylation sites on a region shown to have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Fkh2 protein. The identified motif occurs at region aa692-702 in Fkh2 and has changed constraint in the aligned region aa459-469 in Fkh1. One of the known phosphorylation site in Fkh2 occurs within this region at aa697. Fkh2 and Fkh1 retain their forkhead-associated domain (FHA) and DNA binding domain (DBD). C) The Ace2 protein is known to bind and be phosphorylated by Cbk1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Ace2 protein. The two identified motifs occur at aa121-134 and aa283-287 in Ace2, and changed constraints within the aligned region aa115-128 and aa247-248 (it is a gap) in Swi5. These overlap with the known phosphorylation site in Ace2 (aa122) and the Cbk1 binding site (aa283-286) in Ace2. Both Ace2 and Swi5 retain their DNA binding domain (DBD). Stars represent significant changes in constraints along the lineage. Red double arrow illustrates the duplication event. aa: amino acid position. Scer: S. cerevisiae, Ncas: N. castellii, Zrou: Z. rouxii, Calb: C. albicans.

Mentions: Of these, the paralog pair Rck1/Rck2 contained two predicted motifs that were found to have significant changes in constraints in the Rck1 protein. Interestingly, both motifs are involved in Hog1 signaling [46], [47]. Consistent with our predictions, Rck2 is known to be regulated by Hog1, while Rck1 is thought not to be regulated by Hog1 [47]. However, while our algorithm identified that the motif required for Hog1 binding in Rck2 was evolving more rapidly in Rck1, it is clear that Rck1 preserved some of the critical residues required for binding to Hog1, yet its binding activity to Hog1 has been shown to be poor [47]. This suggests that: 1) the protein ancestral to Rck1/Rck2 is likely to also be regulated by Hog1, and 2) that Rck1 is likely to be regulated in a different manner, having lost or changed critical regulatory sequences after the duplication event (Fig. 4A).


Detecting functional divergence after gene duplication through evolutionary changes in posttranslational regulatory sequences.

Nguyen Ba AN, Strome B, Hua JJ, Desmond J, Gagnon-Arsenault I, Weiss EL, Landry CR, Moses AM - PLoS Comput. Biol. (2014)

Examples of known regulatory motifs with changes in constraints.Alignment of the short linear motifs with known function (indicated with arrows) and significant changes in constraints (red boxes) after gene duplication from representative species. A) The Rck2 protein is known to bind and be phosphorylated by Hog1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Rck2 protein. The two identified motifs occur at aa519-538 and aa577-591 for Rck2, and changed constraints within the aligned region aa439-456 and aa492-506 in Rck1. These overlap with the known phosphorylation site in Rck2 (aa520) and the MAP kinase binding site (aa492-506) in Rck2. Both Rck2 and Rck1 retain kinase function. B) The Fkh2 protein is known to be phosphorylated by Cdk1 at two phosphorylation sites on a region shown to have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Fkh2 protein. The identified motif occurs at region aa692-702 in Fkh2 and has changed constraint in the aligned region aa459-469 in Fkh1. One of the known phosphorylation site in Fkh2 occurs within this region at aa697. Fkh2 and Fkh1 retain their forkhead-associated domain (FHA) and DNA binding domain (DBD). C) The Ace2 protein is known to bind and be phosphorylated by Cbk1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Ace2 protein. The two identified motifs occur at aa121-134 and aa283-287 in Ace2, and changed constraints within the aligned region aa115-128 and aa247-248 (it is a gap) in Swi5. These overlap with the known phosphorylation site in Ace2 (aa122) and the Cbk1 binding site (aa283-286) in Ace2. Both Ace2 and Swi5 retain their DNA binding domain (DBD). Stars represent significant changes in constraints along the lineage. Red double arrow illustrates the duplication event. aa: amino acid position. Scer: S. cerevisiae, Ncas: N. castellii, Zrou: Z. rouxii, Calb: C. albicans.
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pcbi-1003977-g004: Examples of known regulatory motifs with changes in constraints.Alignment of the short linear motifs with known function (indicated with arrows) and significant changes in constraints (red boxes) after gene duplication from representative species. A) The Rck2 protein is known to bind and be phosphorylated by Hog1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Rck2 protein. The two identified motifs occur at aa519-538 and aa577-591 for Rck2, and changed constraints within the aligned region aa439-456 and aa492-506 in Rck1. These overlap with the known phosphorylation site in Rck2 (aa520) and the MAP kinase binding site (aa492-506) in Rck2. Both Rck2 and Rck1 retain kinase function. B) The Fkh2 protein is known to be phosphorylated by Cdk1 at two phosphorylation sites on a region shown to have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Fkh2 protein. The identified motif occurs at region aa692-702 in Fkh2 and has changed constraint in the aligned region aa459-469 in Fkh1. One of the known phosphorylation site in Fkh2 occurs within this region at aa697. Fkh2 and Fkh1 retain their forkhead-associated domain (FHA) and DNA binding domain (DBD). C) The Ace2 protein is known to bind and be phosphorylated by Cbk1 kinase at two motifs that have significant changes in constraints after gene duplication. Numbers indicate residue position within the S. cerevisiae Ace2 protein. The two identified motifs occur at aa121-134 and aa283-287 in Ace2, and changed constraints within the aligned region aa115-128 and aa247-248 (it is a gap) in Swi5. These overlap with the known phosphorylation site in Ace2 (aa122) and the Cbk1 binding site (aa283-286) in Ace2. Both Ace2 and Swi5 retain their DNA binding domain (DBD). Stars represent significant changes in constraints along the lineage. Red double arrow illustrates the duplication event. aa: amino acid position. Scer: S. cerevisiae, Ncas: N. castellii, Zrou: Z. rouxii, Calb: C. albicans.
Mentions: Of these, the paralog pair Rck1/Rck2 contained two predicted motifs that were found to have significant changes in constraints in the Rck1 protein. Interestingly, both motifs are involved in Hog1 signaling [46], [47]. Consistent with our predictions, Rck2 is known to be regulated by Hog1, while Rck1 is thought not to be regulated by Hog1 [47]. However, while our algorithm identified that the motif required for Hog1 binding in Rck2 was evolving more rapidly in Rck1, it is clear that Rck1 preserved some of the critical residues required for binding to Hog1, yet its binding activity to Hog1 has been shown to be poor [47]. This suggests that: 1) the protein ancestral to Rck1/Rck2 is likely to also be regulated by Hog1, and 2) that Rck1 is likely to be regulated in a different manner, having lost or changed critical regulatory sequences after the duplication event (Fig. 4A).

Bottom Line: 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.Our analysis suggests that changes in posttranslational regulation mediated by short regulatory motifs systematically contribute to functional divergence after gene duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Systems Biology, University of Toronto, Toronto, Canada; Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Canada.

ABSTRACT
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 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.

Show MeSH