Teunis J. P. van Dam

The Vertebrate Mitotic Checkpoint Protein BUBR1 Is an Unusual Pseudokinase

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase.

Evolution of modular intraflagellar transport from a coatomer-like progenitor

The intraflagellar transport (IFT) complex is an integral component of the cilium, a quintessential organelle of the eukaryotic cell. The IFT system consists of three subcomplexes [i.e., intraflagellar transport (IFT)-A, IFT-B, and the BBSome], which together transport proteins and other molecules along the cilium. IFT dysfunction results in diseases collectively called ciliopathies. It has been proposed that the IFT complexes originated from vesicle coats similar to coat protein complex (COP) I, COPII, and clathrin. Here we provide phylogenetic evidence for common ancestry of IFT subunits and α, β′, and ε subunits of COPI, and trace the origins of the IFT-A, IFT-B, and the BBSome subcomplexes. We find that IFT-A and the BBSome likely arose from an IFT-B–like complex by intracomplex subunit duplication. The distribution of IFT proteins across eukaryotes identifies the BBSome as a frequently lost, modular component of the IFT. Significantly, loss of the BBSome from a taxon is a frequent precursor to complete cilium loss in related taxa. Given the inferred late origin of the BBSome in cilium evolution and its frequent loss, the IFT complex behaves as a “last-in, first-out” system. The protocoatomer origin of the IFT complex corroborates involvement of IFT components in vesicle transport. Expansion of IFT subunits by duplication and their subsequent independent loss supports the idea of modularity and structural independence of the IFT subcomplexes.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin–proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium.

The multinational SYSCILIA consortium aims to gain a mechanistic understanding of the cilium. We utilize multiple parallel high-throughput (HTP) initiatives to develop predictive models of relationships between complex genotypes and variable phenotypes of ciliopathies. The models generated are only as good as the wet laboratory data fed into them. It is therefore essential to orchestrate a well-annotated and high-confidence dataset to be able to assess the quality of any HTP dataset. Here, we present the inaugural SYSCILIA gold standard of known ciliary components as a public resource.

Evolution of the TOR pathway.

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes.

Protein Complex Evolution Does Not Involve Extensive Network Rewiring

The formation of proteins into stable protein complexes plays a fundamental role in the operation of the cell. The study of the degree of evolutionary conservation of protein complexes between species and the evolution of protein-protein interactions has been hampered by lack of comprehensive coverage of the high-throughput (HTP) technologies that measure the interactome. We show that new high-throughput datasets on protein co-purification in yeast have a substantially lower false negative rate than previous datasets when compared to known complexes. These datasets are therefore more suitable to estimate the conservation of protein complex membership than hitherto possible. We perform comparative genomics between curated protein complexes from human and the HTP data in Saccharomyces cerevisiae to study the evolution of co-complex memberships. This analysis revealed that out of the 5,960 protein pairs that are part of the same complex in human, 2,216 are absent because both proteins lack an ortholog in S. cerevisiae, while for 1,828 the co-complex membership is disrupted because one of the two proteins lacks an ortholog. For the remaining 1,916 protein pairs, only 10% were never co-purified in the large-scale experiments. This implies a conservation level of co-complex membership of 90% when the genes coding for the protein pairs that participate in the same protein complex are also conserved. We conclude that the evolutionary dynamics of protein complexes are, by and large, not the result of network rewiring (i.e. acquisition or loss of co-complex memberships), but mainly due to genomic acquisition or loss of genes coding for subunits. We thus reveal evidence for the tight interrelation of genomic and network evolution.

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome.

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel–Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral–facial–digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.

Phylogeny of the CDC25 homology domain reveals rapid differentiation of Ras pathways between early animals and fungi.

The members of the Ras-like superfamily of small GTP-binding proteins are molecular switches that are in general regulated in time and space by guanine nucleotide exchange factors and GTPase activating proteins. The Ras-like G-proteins Ras, Rap and Ral are regulated by a variety of guanine nucleotide exchange factors that are characterized by a CDC25 homology domain. Here we study the evolution of the Ras pathway by determining the evolutionary history of CDC25 homology domain coding sequences. We identified CDC25 homology domain coding sequences in animals, fungi and a wide range of protists, but not in plants. This suggests that the CDC25 homology domain originated in or before the last eukaryotic ancestor but was subsequently lost in plant. We provide evidence that at least seven different ancestral Ras guanine nucleotide exchange factors were present in the ancestor of fungi and animals. Differences between present day fungi and animals are the result of loss of ancestral Ras guanine nucleotide exchange factors early in fungal and animal evolution combined with lineage specific duplications and domain acquisitions. In addition, we identify Ral guanine exchange factors and Ral in early diverged fungi, dating the origin of Ral signaling back to before the divergence of animals and fungi. We conclude that the Ras signaling pathway evolved by gradual change as well as through differential sampling of the ancestral CDC25 homology domain repertoire by both fungi and animals. Finally, a comparison of the domain composition of the Ras guanine nucleotide exchange factors shows that domain addition and diversification occurred both prior to and after the fungal-animal split.

KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

BackgroundJoubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures.ResultsUsing autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556-/- null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions.ConclusionsWe have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.

OFIP/KIAA0753 forms a complex with OFD1 and FOR20 at pericentriolar satellites and centrosomes and is mutated in one individual with oral-facial-digital syndrome

Oral-facial-digital (OFD) syndromes are rare heterogeneous disorders characterized by the association of abnormalities of the face, the oral cavity and the extremities, some due to mutations in proteins of the transition zone of the primary cilia or the closely associated distal end of centrioles. These two structures are essential for the formation of functional cilia, and for signaling events during development. We report here causal compound heterozygous mutations of KIAA0753/OFIP in a patient with an OFD VI syndrome. We show that the KIAA0753/OFIP protein, whose sequence is conserved in ciliated species, associates with centrosome/centriole and pericentriolar satellites in human cells and forms a complex with FOR20 and OFD1. The decreased expression of any component of this ternary complex in RPE1 cells causes a defective recruitment onto centrosomes and satellites. The OFD KIAA0753/OFIP mutant loses its capacity to interact with FOR20 and OFD1, which may be the molecular basis of the defect. We also show that KIAA0753/OFIP has microtubule-stabilizing activity. OFD1 and FOR20 are known to regulate the integrity of the centriole distal end, confirming that this structural element is a target of importance for pathogenic mutations in ciliopathies.