ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module composed of ARMC9-TOGARAM1-CCDC66-CEP104- CSPP1. TOGARAM1-variants cause JBTS and disrupt its interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and glutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in both ARMC9 and TOGARAM1 patient cells lines suggest a role for this new JBTS-associated protein complex in ciliary stability

A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease

Missense mutations in the WD40 domain of AHI1 cause non-syndromic retinitis pigmentosa

Background: Recent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). Methods: Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. Results: In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1, with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. Conclusions: This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

Tiina Paunio on työryhmän UK10K jäsen.The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.Peer reviewe

Analysis of the <i>CEP290</i> cryptic exons.

<p>(A) Expected transcripts for each model. (B) Exon Y is expressed in all tissues in either <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice. (C) Only in the <i>Cep290</i>^{<i>lca/lca</i>} model, exon X is expressed, either with exon Y or without, at very low levels. The arrow indicates the band that contains both cryptic exons (exon X and exon Y) in the same transcript. <i>Cep290</i>^lca/lca PCR products were run longer to clearly separate the two different bands. (D) Schematic representation of the different transcripts containing cryptic exons found in the three mouse models. Mouse exons and introns are depicted in black, while human exons and introns are represented in blue. Cryptic exons X and Y are shown in grey and red, respectively. Arrows and letters indicate the position of the oligonucleotides in the cryptic exons. Semi-quantification was performed using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#B21" target="_blank">21</a>]. MQ: H₂O (negative control); F: human LCA fibroblasts; B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Analysis of the cryptic exons X and Y in human retina and fibroblast cell lines.

<p>The expression of the <i>CEP290</i> human exons 26 and 27, and the cryptic exons Y and exon X were assessed in tissues derived from both mice and humans. Actin was used for normalization. </p

Transcriptional characterisation of <i>Cep290</i> in humanized mouse models.

<p>(A-B) <i>Cep290</i> expression levels in various mouse tissues were assessed by RT-PCR. Regions containing murine exons 10 to 13 (A) and 24 to 27 (B) were analyzed. No differences were observed. (C) Amplification using human primers was also assessed in the three models. Only humanized models showed amplification. (D) Actin was used for normalization and comparison among tissues and models. MQ: H₂O (negative control); B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Morphological and CEP290 immunolocalization analyses in P150 mouse retinas.

<p>Immunodetection of CEP290 and acetylated tubulin in retinal sections from <i>Cep290</i>^<i>wt/wt</i>, <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice, at P150. Images were taken at 200X (A) and 400x (B) magnifications. CEP290 (green) is located in the connecting cilium (CC) and remains unaltered in the humanized models. Nuclei were marked with DAPI (blue) and CC with acetylated tubulin (red). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PhL: photoreceptor layer; RPE: retinal pigment epithelium; IS: inner segment; CC: connecting cilium; OS: outer segment.</p