A unied nomenclature for vertebrate olfactory receptors Tsviya Olender ( [email protected] ) The Weizmann Institute of Science https://orcid.org/0000-0002-4194-6420 Tamsin E.M. Jones European Molecular Biology Laboratory Michal Twik Weizmann Institute of Science Elspeth Bruford European Molecular Biology Laboratory Doron Lancet Weizmann Institute of Science Methodology article Keywords: Olfaction, Nomenclature, Olfactory receptors, Orthologs, Paralogs, Evolution Posted Date: September 24th, 2019 DOI: https://doi.org/10.21203/rs.2.14887/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Evolutionary Biology on April 15th, 2020. See the published version at https://doi.org/10.1186/s12862-020-01607-6. Page 1/20 Abstract Background Olfactory receptors (ORs) are G protein-coupled receptors with a crucial role in odor detection. A typical mammalian genome harbors ~1000 OR genes and pseudogenes; however, different gene duplication/deletion events have occurred in each species, resulting in complex orthology relationships. While the human OR nomenclature is widely accepted and based on phylogenetic classication into 18 families and further into subfamilies, for other mammals different and multiple nomenclature systems are currently in use, thus concealing important evolutionary and functional insights. Results Here we describe the Mutual Maximum Similarity (MMS) algorithm, a systematic classier for assigning a human-centric nomenclature to any OR gene based on inter-species hierarchical pairwise similarities. MMS was applied to the OR repertoires of seven mammals and zebrash. Altogether, we assigned symbols to 10,249 ORs. This nomenclature is supported by both phylogenetic and synteny analyses. The availability of a unied nomenclature provides a framework for diverse studies, where textual symbol comparison allows immediate identication of potential ortholog groups as well as species-specic expansions/deletions; for example, Or52e5 and Or52e5b represent a rat- specic duplication of OR52E5 . Another example is the complete absence of OR subfamily OR6Z among primate OR symbols. In other mammals, OR6Z members are located in one genomic cluster, suggesting a large deletion in the great ape lineage. An additional 14 mammalian OR subfamilies are missing from the primate genomes. While in chimpanzee 87% of the symbols were identical to human symbols, this number decreased to ~50% in dog and cow and to ~30% in rodents, reecting the adaptive changes of the OR gene superfamily across diverse ecological niches. Application of the proposed nomenclature to zebrash revealed similarity to mammalian ORs that could not be detected from the current zebrash olfactory receptor gene nomenclature. Conclusions We have consolidated a unied standard nomenclature system for the vertebrate OR superfamily. The new nomenclature system will be applied to cow, horse, dog and chimpanzee by the Vertebrate Gene Nomenclature Committee and its implementation is currently under consideration by other relevant species-specic nomenclature committees. Background Olfactory receptors (ORs) are G protein-coupled receptors with an essential role in odor detection. Being the largest gene family in vertebrates, a typical mammalian genome harbors ~1000 OR genes and pseudogenes; however, the numbers of functional OR genes vary enormously among genomes of different animals, reecting the adaptation of organisms to different environments [1-4]. ORs are distributed in clusters on most mammalian chromosomes. There are strong indications of a common ancestry for most OR clusters [5], a feature which might be related to common cis-regulatory elements [6, 7]. Nevertheless, processes of gene duplication and gene deletion have taken place in each species [1-4], making orthology relationships dicult to determine [8], and hence requiring careful manual curation for the assignment of gene nomenclature. Another complexity arises from the high content of OR pseudogenes in some organisms, such as human (55% pseudogenes) [9]. Thus, assigning orthology- Page 2/20 based symbols to the OR gene superfamily is challenging and requires conceptual translation of all OR pseudogenes. For many years, an ocial nomenclature system has been in place for human ORs [10] that is widely accepted and utilized by the community. The human nomenclature is based on a sequence similarity classication of the OR repertoire into 18 families and > 300 subfamilies, where symbols consist of the root “OR” followed by a family numeral, subfamily letter(s), and a numeral representing the individual gene within the subfamily. For example, OR3A1 is member 1 of family 3, subfamily A, and OR7E12P is an OR pseudogene that is member 12 of family 7, subfamily E. The details of the classication method were previously described [9-12]. Briey, a new gene is classied into the same subfamily if it shows at least 60% protein sequence identity to the best hit; family membership is based on at least 40% sequence identity at the protein level. This nomenclature has already been applied to dog, platypus and opossum [5, 13, 14] and is available to the community via a dedicated database, the Human Olfactory Receptor Data Explorer (HORDE) [11, 15, 16]. In some other vertebrates, different nomenclature systems are currently in use. For example, several nomenclature systems have previously been published for mouse. One utilized the prex 'MOR', followed by a family number, a hyphen (-) and a number representing the individual gene within the family, with 'P' at the end to denote a pseudogene (e.g. MOR1-1 and MOR185-9P) [17]; another was based on genomic location [18]. The current Mouse Genome Informatics (MGI) ocial nomenclature uses the root ‘Olfr’ followed by a number, with ‘-ps’ and another number at the end for pseudogenes (e.g. Olfr562 and Olfr1001-ps1). This serial number-based nomenclature conceals important structural and functional insights. Moreover, it deviates from the rule applied to almost all other genes, whereby, when possible, the symbols for orthologous genes in human and other vertebrates should be identical (excluding capitalization). Another example of deviation from this rule is the zebrash OR nomenclature, which is based on a phylogenetic classication into subfamilies with sequential numbers starting from 101 (e.g. or101-1 and or102-1 are two genes in two different subfamilies) [19]. As sh ORs are highly diverged [20], the designated OR subfamilies in sh are expected to be lineage-specic and, therefore, this nomenclature [19] is not useful to detect similarities across sh and vertebrates. The use of different nomenclature in different organisms creates diculties both when comparing genes across species and, especially, when the same gene is reported more than once under different names. Although this situation is also common with other genes, it becomes especially confusing in large gene families that are found in multiple species, such as the ORs. Next generation technologies are dramatically increasing the number of sequenced vertebrates. Therefore, the availability of a unied and widely accepted nomenclature that encodes homology relationships becomes more important than ever. Here, we propose a unied nomenclature system for vertebrate OR genes and pseudogenes. The nomenclature is human-centric and therefore based on the human classication system for OR genes. Using a dedicated algorithm (Mutual Maximum Similarity, MMS), we applied our nomenclature system to the OR repertoires of mouse, rat, cow, dog, horse, orangutan and chimpanzee and, nally, also to Page 3/20 zebrash, a more distantly related vertebrate species. We show that the nomenclature captures the phylogenetic relationships among the studied species and provides a powerful framework for diverse studies of vertebrate ORs. A unied nomenclature for the OR gene family can also serve as a model for other large multigene families, allowing researchers to easily make cross-species comparisons in complex groups of genes. All of the nomenclature data are available from the HORDE database (https://genome.weizmann.ac.il/horde/), and are under consideration by the relevant species-specic nomenclature committees that are using an alternative OR nomenclature, namely MGNC for mouse [21], RGNC for rat [22] and ZNC for zebrash [23]. The Vertebrate Gene Nomenclature Committee (VGNC) [24] are currently naming genes within chimpanzee, cow, dog and horse and will adopt this OR nomenclature in these species. Results The MMS algorithm The MMS algorithm assigns human-based OR symbols by detecting the inter-species hierarchical pairwise similarities (Fig. 1). The algorithm rst analyzes the all-versus-all BLASTP identity matrix of the given OR repertoire versus human. Mutual best hits with 80% identity are rst identied and are assigned the same symbol as the human best hit, second-best ortholog candidates with 80% identity are then identied and are assigned with the human best hit gene symbol with the addition of the letters B, C, etc. For example, OR9A4 is the mutual best hit and OR9A4B is the second best hit. The remaining ORs are compared to non-human ORs to detect non-human OR orthology relationships. OR genes that have not already been named in the above steps are classied into families and subfamilies as shown in Fig. 1 and as explained in [11], either as a new member of a subfamily with the n+1 subfamily member number, or classied into