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, 34 (8), 2016-2034

Insights Into Ciliary Genes and Evolution From Multi-Level Phylogenetic Profiling

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Insights Into Ciliary Genes and Evolution From Multi-Level Phylogenetic Profiling

Yannis Nevers et al. Mol Biol Evol.

Abstract

Cilia (flagella) are important eukaryotic organelles, present in the Last Eukaryotic Common Ancestor, and are involved in cell motility and integration of extracellular signals. Ciliary dysfunction causes a class of genetic diseases, known as ciliopathies, however current knowledge of the underlying mechanisms is still limited and a better characterization of genes is needed. As cilia have been lost independently several times during evolution and they are subject to important functional variation between species, ciliary genes can be investigated through comparative genomics. We performed phylogenetic profiling by predicting orthologs of human protein-coding genes in 100 eukaryotic species. The analysis integrated three independent methods to predict a consensus set of 274 ciliary genes, including 87 new promising candidates. A fine-grained analysis of the phylogenetic profiles allowed a partitioning of ciliary genes into modules with distinct evolutionary histories and ciliary functions (assembly, movement, centriole, etc.) and thus propagation of potential annotations to previously undocumented genes. The cilia/basal body localization was experimentally confirmed for five of these previously unannotated proteins (LRRC23, LRRC34, TEX9, WDR27, and BIVM), validating the relevance of our approach. Furthermore, our multi-level analysis sheds light on the core gene sets retained in gamete-only flagellates or Ecdysozoa for instance. By combining gene-centric and species-oriented analyses, this work reveals new ciliary and ciliopathy gene candidates and provides clues about the evolution of ciliary processes in the eukaryotic domain. Additionally, the positive and negative reference gene sets and the phylogenetic profile of human genes constructed during this study can be exploited in future work.

Keywords: ciliopathies; cilium; comparative genomics; evolution; phylogenetic profiling.

Figures

<sc>Fig</sc>. 1.
Fig. 1.
Schematic representation of cilium. Structural components of a cilium and intraflagellar-transport machinery. The basal body is anchored at the membrane and is the basis of the axoneme. The transition zone filters the molecules that enter and leave the cilium, allowing maintenance of the organelle integrity. IFT-particles mediate the intracellular transport: IFT-B linked to kinesin in the anterograde direction, IFT-A and dynein in the retrograde direction. The cross-sections detail ultrastructural differences between primary and motile cilium, notably the molecular machinery allowing the movement for motile cilium.
<sc>Fig</sc>. 2
Fig. 2
Average phylogenetic profiles of each hierarchical cluster. Species names are colored according to major eukaryotic clades (from bottom to top: Excavata, Plants, Stramenopiles, Alveolata, Amoebozoa, Fungi, Holozoa, i.e., Metazoa, and their closest single-celled relatives). Each column corresponds to a cluster, ranked according to the taxonomical rank to which they correspond starting from human specific (cluster1) to “universal” eukaryotic genes (Cluster 9 and 10). Cluster from 11 to 14 do not correspond to a specific taxonomical division and are ordered by descending size. The circle size is proportional to the percentage of genes from a given cluster with an ortholog found in a given species. The far right column “Cilium” indicates ciliated species (full green circle) and nonciliated species (empty circle). Species for which the existence of a ciliated state was unclear have no circle. Distribution of the Cluster 14 correlates to cilium distribution. This figure was generated using the iTol website (Letunic and Bork 2011).
<sc>Fig</sc>. 3.
Fig. 3.
Venn diagram of the genes predicted by three methods. Number of genes in each subcategory is indicated in black. True positives and false positives are indicated in green and red, respectively.
<sc>Fig</sc>. 4.
Fig. 4.
Venn diagram of the genes predicted by this work and three previous studies. Number of genes in each subcategory is indicated in black. True positives and false positives are indicated in green and red, respectively. The corresponding enrichment is indicated by the hypergeometric P value. In the original work, genes predicted in Dey et al. (2015) were collapsed in orthology groups, with no distinction between paralogs. The numbers between parentheses correspond to the numbers presented in the original publications: the number of groups of genes predicted. The number of individual genes and true positives present in those groups is indicated with no parentheses and was used to determine overlap.
<sc>Fig</sc>. 5.
Fig. 5.
Hierarchical clustering dendrogram of ciliary gene profiles in eukaryotic species. Homogeneous clusters are collapsed. The complete dendrogram is available in supplementary material, Supplementary Material online. Approximately Unbiased (AU) P value of each branch is annotated in red. Ciliated species and collapsed clusters with exclusively ciliated species are colored in green, nonciliated species and clusters in red. Species for which the ciliated state is unclear are in black. Significant clusters (AU ≥ 0.90) are framed by a red rectangle and annotated by a greek letter. Collapsed cluster are homogeneous in term of presence of cilia, and are annotated with the number of species they contain, and the minimal and maximal number of orthologs detected in these species. The cluster δ contains both ciliated and nonciliated species. It forks in two subclusters (separated by a dotted line): subcluster a corresponds to nematodes and ticks, representative of Ecdysozoa and subcluster b to a mixed group of eukaryotic species.
<sc>Fig</sc>. 6.
Fig. 6.
Evolutionary modules based on divergence in metazoan phylogenetic profiles.Partition of phylogenetic profiles of the 274 ACG into four evolutionary modules. Species names are colored according to major eukaryotic clades (from left to right: Excavates, Plants, Stramenopiles, Alveolates, Amoebozoa, Fungi, and Holozoa). Ciliated species are annotated with a full green circle and nonciliated with an empty circle. Species for which existence of a ciliated stage is unclear have no circle. 274 binary profiles of presence (full colored rectangle) and absence (blank) of proteins colored according to their evolutionary modules: blue for proteins conserved in both nematodes and insects, orange for absence in all nematodes and presence in most insects, gray for presence in nematodes but absence in all insects (eight genes), purple for the rest (lost in most Ecdysozoa). This figure was generated using the iTol website (Letunic and Bork 2011).
<sc>Fig</sc>. 7.
Fig. 7.
Ciliary localization of five predicted ciliary proteins. Immunofluorescence of predicted ciliary proteins in ciliated mammalian cells. The cilia are labeled with antibodies targeting the ciliary markers ARL13B or acetylated tubulin (Ac Tub). (AD) LRRC34, LRRC23, TEX9, and WDR27 colocalize with cilia in HK2 cells (arrowheads). (E) The mouse ortholog of BIVM localizes to the base of cilia (basal body and centriole, arrowheads) in mIMCD3 cells. Insets show a magnified view of a single cilium taken from an independent field of view.

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