Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Abstract

1Despite over a billion years of evolutionary divergence, several thousand human genes possess 2 clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in 3 one or both lineages. The ortholog conjecture postulates that orthologous genes between species 4 retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free 5 to diverge in function. However, the retention of ancestral functions among co-orthologs between 6 species and within gene families has been difficult to test experimentally at scale. In order to 7 investigate how ancestral functions are retained or lost post-duplication, we systematically 8 replaced hundreds of essential yeast genes with their human orthologs from gene families that have 9 undergone lineage-specific duplications, including those with single duplications (one yeast gene 10 to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a 11 variable pattern of replaceability across different ortholog classes, with an obvious trend towards 12 differential replaceability inside gene families, rarely observing replaceability by all members of 13 a family. We quantify the ability of various properties of the orthologs to predict replaceability, 14showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and 15 tissue-specific expression of the human co-orthologs, i.e. the human proteins that are less diverged 16 from their yeast counterpart and more ubiquitously expressed across human tissues more often 17 replace their single yeast ortholog. These trends were consistent with in silico simulations 18demonstrating that when only one ortholog is replaceable, it tends to be the least diverged of the 19 pair. Replaceability of yeast genes having more than two human co-orthologs was marked by 20 retention of orthologous interactions in functional or protein networks as well as by more ancestral 21 subcellular localization. Overall, we performed >400 human gene replaceability assays revealing 22 56 new human-yeast complementation pairs, thus opening up avenues to further functionally 23 characterize these human genes in a simplified organismal context. 24 25 and are distinguished from those related by speciation, termed orthologs [8,9]. Importantly, an 46 expanded family of paralogs in one lineage may be co-orthologs to a single gene in another lineage. 47How ancestral functions are partitioned, lost, or retained between paralogs and orthologs during 48 these duplication events is a major topic of study for evolutionary biology. To address these 49 questions, the ortholog conjecture was put forth, a major thesis stating that orthologs are more 50 likely to retain function between species than paralogs, which will tend to diverge in function after 51 duplication due to drift and relaxed selection [10,11]. This conjecture underlies common methods 52 by which functions are assigned to newly discovered or understudied genes in un-annotated 53 4 genomes ...

The Ortholog Conjecture Revisited: the Value of Orthologs and Paralogs in Function Prediction

“…[49]) and for some types of genes [1], the higher similarity of orthologs in some newer datasets was accompanied by either no change in functional similarity over time [2,5] or the increase in functional similarity over time [43]. As a decrease in structural [41,37] and functional [23,35,34,4,9,32] similarity with divergence is a widely expected and observed pattern for both paralogs and orthologs, the patterns of evolution in these studies are indeed baffling. Further examination of several of these studies has uncovered problems with the analyses such that there is either no longer support for the ortholog conjecture [15] or that there was no statistical support for the ortholog conjecture in the first place, as in the case of human-mouse comparisons in Ref.…”

Systematic humanization of the yeast cytoskeleton discerns functionally replaceable from divergent human genes

“…Cross-species gene swaps provide an opportunity to directly test the functional divergence of orthologous genes even over large evolutionary timescales. While Saccharomyces cerevisiae (Baker’s yeast) and Homo sapiens (humans) diverged from their opisthokont ancestor nearly a billion years ago, both species still share thousands of orthologous genes and high-throughput humanization assays in yeast have found that many human genes are capable of substituting for their yeast orthologs with rates up to 47%, depending on strains and assays 5–7, 9–11 . Most of these complementation tests until recently were performed in the absence of gene family expansions and revealed many humanizable systems, including the proteasome, and sterol and heme biosynthesis pathways 5–9, 11 .…”

“…While Saccharomyces cerevisiae (Baker’s yeast) and Homo sapiens (humans) diverged from their opisthokont ancestor nearly a billion years ago, both species still share thousands of orthologous genes and high-throughput humanization assays in yeast have found that many human genes are capable of substituting for their yeast orthologs with rates up to 47%, depending on strains and assays 5–7, 9–11 . Most of these complementation tests until recently were performed in the absence of gene family expansions and revealed many humanizable systems, including the proteasome, and sterol and heme biosynthesis pathways 5–9, 11 . In this study, we sought to better understand the functional equivalence of human and yeast orthologs that play key structural roles in the eukaryotic cytoskeleton, particularly focusing on how gene family expansions in cytoskeletal lineages might have diversified in function across their respective gene families.…”

“…However, only recently have efforts been made to test such functional equivalence more systematically, with several recent large-scale studies harnessing “the awesome power of yeast genetics” to systematically replace yeast genes by their human, plant, or even bacterial counterparts and assay for functional compatibility 5–10 . Although humans and yeast last shared a common ancestor nearly a billion years ago, these studies have demonstrated that substantial fractions (12-47%) of tested essential yeast genes could be replaced by their human equivalents 5–9, 11 . The ability of many human genes to functionally replace their yeast orthologs demonstrates the high degree of functional conservation in eukaryotic systems over billion year evolutionary timescales 9, 10 .…”

“…Previous humanization efforts in yeast have primarily focused on ortholog pairs with no obvious duplications within yeast and human lineages (1:1 orthologs), only partially testing the orthologs in expanded gene families 5–7, 9, 11 and seldom beyond assaying impact on growth rate. In this study, to better understand functional conservation across expanded gene families in core eukaryotic processes, we focused on the major structural components of the eukaryotic cytoskeleton, including actins, myosins, septins, and tubulin genes.…”