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Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes

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Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878751; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Systematic humanization of the yeast cytoskeleton discerns functionally replaceable from divergent human genes Riddhiman K. Garge1, Jon M. Laurent1,2, Aashiq H. Kachroo3,*, Edward M. Marcotte1,* 1Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, TX, USA 2Institute of Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, NY, USA 3The Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montreal, QC, Canada *For correspondence: [email protected], [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878751; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Abstract 2 3 Many gene families have been expanded by gene duplications along the human lineage, relative 4 to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is 5 understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular 6 processes including chromosome segregation, macromolecular transport, and cell shape 7 maintenance. To determine functional redundancy and divergence of duplicated human genes, we 8 systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ~85% of 9 human cytoskeletal genes across 7 gene families for their ability to complement a growth defect 10 induced by deletion of the corresponding yeast ortholog. In 5 of 7 families—all but α-tubulin and 11 light myosin, we found at least one human gene capable of complementing loss of the yeast gene. 12 Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell 13 morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with 14 deletion phenotypes of their interaction partners, we identify instances of human genes in the actin 15 and septin families capable of carrying out essential functions, but apparently failing to interact 16 with components of the yeast cytoskeleton, thus leading to abnormal cell morphologies. Overall, 17 we show that duplicated human cytoskeletal genes appear to have diverged such that only a few 18 human genes within each family are capable of replacing the essential roles of their yeast orthologs. 19 The resulting yeast strains with humanized cytoskeletal components now provide surrogate 20 platforms to characterize human genes in simplified eukaryotic contexts. bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878751; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 21 Introduction 22 23 Gene duplication is regarded as one of the key drivers of evolution, contributing to the generation 24 and accumulation of new genetic material within species1,2. Duplication creates an initial 25 multiplication of dosage and functional redundancy, but the trends dictating how duplicated genes 26 retain function or diverge are still unclear. The processes governing the distribution of molecular 27 roles within gene families also have important consequences for annotating genes, which generally 28 takes advantage of sequence similarity and conservation over vast timescales of divergence to infer 29 functions of homologous genes across species. Many individual studies have directly tested the 30 conservation of function among orthologs from different species by swapping them from one 31 species into another3,4. However, only recently have efforts been made to test such functional 32 equivalence more systematically, with several recent large-scale studies harnessing “the awesome 33 power of yeast genetics” to systematically replace yeast genes by their human, plant, or even 34 bacterial counterparts and assay for functional compatibility5–10. Although humans and yeast last 35 shared a common ancestor nearly a billion years ago, these studies have demonstrated that 36 substantial fractions (12-47%) of tested essential yeast genes could be replaced by their human 37 equivalents5–9,11. The ability of many human genes to functionally replace their yeast orthologs 38 demonstrates the high degree of functional conservation in eukaryotic systems over billion year 39 evolutionary timescales9,10. 40 41 Previous humanization efforts in yeast have primarily focused on ortholog pairs with no obvious 42 duplications within yeast and human lineages (1:1 orthologs), only partially testing the orthologs 43 in expanded gene families5–7,9,11 and seldom beyond assaying impact on growth rate. In this study, 44 to better understand functional conservation across expanded gene families in core eukaryotic 45 processes, we focused on the major structural components of the eukaryotic cytoskeleton, 46 including actins, myosins, septins, and tubulin genes. Genes constituting the eukaryotic 47 cytoskeleton play key roles in critical cellular processes, mainly organizing the contents of the cell 48 by dynamically controlling cell shape, positioning organelles, and transporting macromolecules 49 including chromosomes across the cell through the generation of coordinated mechanical forces12– 50 14. Importantly, cytoskeletal gene families have undergone large expansions along the human 51 lineage, while being restricted to only a few family members in yeast (Fig. 1). bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878751; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 52 53 Advances in comparative genomics have shed light on the likely cytoskeletal components present 54 in the last eukaryotic common ancestor (LECA)12–14. Additionally, phylogenetic profiling studies 55 of the cytoskeleton have been highly informative in inferring gene loss and retention events across 56 eukaryotic clades13,14 (Fig. 1). Such studies suggest that the origins of the eukaryotic cytoskeleton 57 predate eukaryogenesis and ancestrally trace back to primitive tubulin- and actin-like homologs in 58 bacteria13. These components critical in cell division subsequently evolved to incorporate families 59 of accessory motors and regulatory proteins expanding towards performing vital cellular roles, 60 including phagocytosis, motility and vesicular transport, still evident across vast eukaryotic clades 61 of life13,15–18. 62 63 Though the cellular roles of human cytoskeletal gene families have been broadly elucidated, aided 64 by studies in simpler eukaryotes19–23, the specific functions of their constituent family members in 65 humans have to date still only been partially characterized24–30. Functional assays in human cell 66 lines pose the challenge of functional redundancy, with buffering by other paralogs complicating 67 the determination of paralog-specific roles within cytoskeletal gene families. The high degree of 68 sequence conservation among paralogs within each cytoskeletal family make functional analysis 69 of individual cytoskeletal genes directly in human cells both experimentally and computationally 70 cumbersome. However, cross-species gene swaps have the potential to provide direct assays of 71 individual paralogs within these expanded gene families, thereby revealing the extent to which 72 present day orthologous genes retain ancestral function. 73 74 To understand the extent to which human cytoskeletal genes in expanded orthogroups retain cross- 75 species functional equivalence, we systematically humanized major elements of the yeast 76 cytoskeleton. We tested ~85% (50/59) of all human genes from actin, myosin, septin, and tubulin 77 families, using a combination of classical yeast genetics and CRISPR-Cas9 mediated genome 78 editing to assay the replaceability of essential cytoskeletal orthologs, as initially determined via 79 simple growth rescue complementation assays. Overall, we show that (13/59) members from 5 of 80 7 tested gene families (actin, heavy myosin, septin, β- and ɣ-tubulin) can indeed execute essential 81 roles of their yeast counterparts. Within each replaceable family we show that several present-day 82 human orthologs still possess functional roles of their respective opisthokont ancestors compatible bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878751; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 83 in a yeast cellular context. Additionally, we characterized cellular phenotypes beyond growth and 84 observed differential abilities among complementing
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