CEP290 protein analysis in <i>Cep290</i> mouse models.

<p>Representative CEP290 immunodetection in P150 retinas from the three different mouse models. A CEP290-Flag construct was expressed in HEK293-T cells and used as a positive control. Tubulin was used for normalization.</p

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

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

Generation of the <i>Cep290</i> humanized mouse models.

<p>(A) Structure of the <i>CEP290</i> gene in human and mouse (drawn to scale). (B) Two mouse models were generated by introducing human exons 26 and 27 and intron 26, either with or without the LCA-causing mutation (depicted with *), to the mouse <i>Cep290</i> gene. Human and mouse loci, targeting vector and recombinant locus are depicted. Arrows and letters indicate the position of the oligonucleotides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#pone.0079369.s001" target="_blank">Table S1</a>). </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

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

MOESM1 of Cellular ciliary phenotyping indicates pathogenicity of novel variants in IFT140 and confirms a Mainzer–Saldino syndrome diagnosis

Additional file 1: Figure S1. Schematic overview of the workflow described in this study. Figure S2. Sequence validation of CRISPR/Cas9-derived Ift140 knockout cells

DataSheet1_A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome.DOCX

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.</p

Table2_A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome.XLSX

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.</p

Video2_A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome.AVI

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.</p