Abstract
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.
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Acknowledgements
Our deepest thanks go to all of the families and individuals with Joubert syndrome, Jeune syndrome and retinitis pigmentosa. We thank P. Barker, M. Jackson, K. Roberts and A. Jones from PerkinElmer Inc. for technical support. The research received funding from the European Community’s Seventh Framework Programme FP7/2009 under grant agreement no: 241955 SYSCILIA towards C.A.J., R.R., M.U., T.G., P.L.B., O.E.B., U.W., R.H.G., M.A.H. and H.O. This work was supported by a Sir Jules Thorn Award for Biomedical Research (JTA/09, to C.F.I. and C.A.J.) and a Medical Research Council grant (MR/K011154/1, to C.A.J.). M.S. was financially supported by an Action Medical Research Clinical Training Fellowship (RTF-1411), a Radboud University Excellence fellowship, a Radboud UMC Hypatia Tenure Track fellowship and acknowledges funding from the Deutsche Forschungsgemeinschaft (DFG; grant SFB 1140 (KIDGEM)). R.R. is funded by the Netherlands Organization for Scientific Research (NWO Vici-865.12.005), and by the Foundation Fighting Blindness (C-CMM-0811-0546-RAD02). P.L.B. is a Wellcome Trust Senior Research Fellow, and P.L.B., M.S., R.H.G. and R.R. acknowledge funding from the Dutch Kidney Foundation (CP11.18, ‘Kouncil’). C.T.T. is financially supported by DFG grant TH 896/3-3 and IZKF (Interdisciplinary Centre for Clinical Research of the Universität of Erlangen-Nürnberg) grant F4. U.W. is financially supported by the FAUN foundation, Nuernberg. Z.A.A. and W.H. are supported by grants from the Rosetree’s Trust (A210 and A465). H.M.M. is supported by the Great Ormond Street Hospital Children’s Charity and received grants from the Milena Carvajal Pro-Kartagener Foundation and Action Medical Research (GN2101). H.R. is financially supported by the Canadian Institutes of Health Research (CIHR) Training Program in Genetics, Child Development and Health, Alberta Children’s Hospital Research Institute (ACHRI) and Alberta Children’s Hospital Foundation. A.R.W. is financially supported by RP Fighting Blindness, Moorfields Eye Hospital Special Trustees, the National Institute for Health Research (NIHR; Moorfields Eye Hospital and Institute of Ophthalmology, London, UK) and the Foundation Fighting Blindness (US). D.D. is financially supported by NINDS grant R01NS064077, the University of Washington Intellectual and Developmental Disabilities Research Center Genetics Core P30HD002274, and private donations from families of individuals with Joubert syndrome. J.S. is financially supported by NCI grant R21CA160080. Access to the B6;129P2-Tmem67tm1Dgen/H line was financially supported by the Wellcome Trust Knock-out Mouse Resource scheme (C.A.J. and C.F.I.; grant ME041596). The UK10K project is financially supported by the Wellcome Trust under grant agreement WT091310. A full list of UK10k investigators can be accessed at http://www.uk10k.org. The University of Washington Center for Mendelian Genomics is supported by NHGRI grant U54HG006493.
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K.S., G.W., S.N., W.H., M.Adams and J.H. performed the primary and secondary siRNA screens. K.S., G.W., D.A.M., T.-M.T.N. and S.J.F.L. performed and analysed the tertiary and other validation screens. L.W. wrote scripts for UPS validation screen analysis. C.T., S.M.B., J.B., E.E.M., D.C.T. and C.F.I. made substantial contributions to the organization and management of the siRNA and validation screens, and helped draft and edit the manuscript. S.N. performed quantitative RT-PCR analyses, and K.S., G.W. and W.H. carried out western blot analyses in validation screens. G.W. and Z.A.A. performed and interpreted immunofluorescence and immunohistochemical staining of mouse tissue sections. D.A.P. wrote Perl scripts for automated analysis of siRNA screen data and reanalysed UK10k WES data. K.S. and G.W. performed confocal immunofluorescence microscopy of primary fibroblasts and cell lines. G.T. and T.G. developed software and analysed array CGH data, and G.T. analysed siRNA sequences. G.G.S. and R.H.G. performed three-dimensional spheroid cultures of mIMCD3 cells. J.K. and O.E.B. performed and interpreted all experiments with C. elegans. N.S., U.W. and K.A.W. analysed mouse photoreceptor cells by high-magnification immunofluorescence and immunoelectron microscopy. K.K., A.G. and C.T.T. developed and characterized the NEK1 antibody. H.R., C.L.B., P.G., R.T.P., R.L. and F.P.B. ascertained and recruited subjects, performed linkage analysis, WES and Sanger sequencing, and analysed data for Hutterite participants. J.S.P. and A.M.I. diagnosed and enrolled participants, collated clinical documentation and supervised research on Hutterite participants. C.O. analysed and provided data for Schmiedeleut controls. R.H.G. analysed and provided data for Dariusleut and Leherleut controls. L.H. and K.M.B. provided reagents for work on PIBF1. I.G.P. analysed the Joubert syndrome exomes, designed the MIPS capture and performed the MIPS sequencing and analysis. J.S. conceived of the MIPS method and E.A.B. contributed to MIPS method development. D.D. diagnosed and enrolled participants, collated clinical documentation, supervised the sequencing and edited the manuscript. A.M.I., B.N.C., A.E.C., C.E.W., K.M.B., P.L.B., F.S.A., M.M., C.T., C.F.I., H.O., C.E.W., F.S., S.A.H. and P.F. diagnosed and ascertained participants and collated clinical information. A.R.W. diagnosed and ascertained participants, collated clinical information and analysed SNP genotyping data. P.I.S. collated clinical information, prepared samples for WES and analysed WES data. M.Aldahmesh and S.A. performed RT-PCR experiments. T.-M.T.N., D.A.M. and J.v.R. performed and analysed C21orf2 PPI experiments and immunofluorescence microscopy. N.H., K.B. and M.U. performed, analysed and supervised TAP mass spectrometry experiments. M.S. performed WES analysis and zebrafish experiments, performed and supervised Sanger sequencing analysis and analysed the data, and helped draft the manuscript. The co-corresponding authors jointly supervised the work. D.D. diagnosed and enrolled participants, collated clinical documentation, supervised the human genetics experiments, and helped draft and edit the manuscript. H.M.M. conceived the human genetics and zebrafish experiments, supervised the research and wrote the manuscript. R.R. and C.A.J. conceived the study, directed and supervised the research, analysed and collated data, and wrote the manuscript.
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Supplementary Figure 1 Characterization of the antibody specificities and sub-cellular localizations of hit G protein-coupled receptors.
(a) Localization of the indicated GPCRs (green; with grey-scale image for the green channel on the right) for mIMCD3 cells to the ciliary base (white arrowheads). GPCRs include GPR20 (orphan G-protein coupled receptor 20), GPR173 (orphan G-protein coupled receptor 173), CRHR2 (corticotropin releasing hormone receptor 2), HTR1B (5-hydroxytryptamine/serotonin receptor), MAS1 (MAS1 proto-oncogene receptor), OPRL1 (opioid receptor-like 1), P2RY14 (purinergic receptor P2Y, G-protein coupled, 14) and RAPSYN (receptor-associated protein of the synapse). Knock-down with the cognate siRNA pools for all GPCRs caused loss of primary cilia (visualized by acetylated α-tubulin; red), compared to scrambled (scr.) negative control. Scale bar = 5 μm. (b) Localization of GPCRs (green) to the base of primary cilia (stained for polyglutamylated α-tubulin or γ-tubulin as indicated; red) in the different cortical layers of Tmem67+/+ wild-type embryonic (E18.5) mouse neocortex (left panels). In Tmem67−/− knock-out embryos mutated for the transition zone receptor TMEM67, the ciliary localization of GPCRs was lost (right panels). Images are representative of two independent experiments. Scale bar = 20 μm. Magnified regions are shown in the smaller panels.
Supplementary Figure 2 Mutations in PIBF1 cause Joubert syndrome.
Homozygosity mapping and pedigrees for Hutterite families with Joubert syndrome. (a) Plot of SNP genotypes in family H1, showing two large intervals of absence of heterozygosity (red lines) on chromosomes 10 and 13. (b) Pedigrees showing segregation of c.1910A > C in PIBF1 (NM_006346.2) in Hutterite families H1 to H4 with Joubert syndrome. ⋆ indicates individuals with microphthalmia and anterior segment dysgenesis that does not segregate with the homozygous variant. NA, not available. (c) Electropherogram of the homozygous PIBF1 mutation c.1910A > C p.Asp637Ala in affected individual H1-4 compared to a control individual, indicated by red arrowhead. (d) Pedigree and electropherograms for the compound heterozygous PIBF1 variants c.1669delC p.Leu637Phefs*18 and c.1214G > A p.Arg405Gln in affected individual UW155-3 with Joubert syndrome, indicated by red arrowheads. (e) Pedigree of family MTI-121 with compound heterozygous PIBF1 variants c.1214G > A p.Arg405Gln and a genomic deletion encompassing exons 6 to 9 (c.673-?_1322 + ?del) of PIBF1 in affected individuals II.5 and II.7. Electropherogram of the hemizygous c.1910A > C mutation in PIBF1 in affected individual II:2 (lower electropherogram), indicated by the red arrowhead, as compared to a control individual (upper electropherogram).
Supplementary Figure 3 Range of brain MRI findings in individuals with PIBF1-related Joubert syndrome.
(a–c) Mild vermis hypoplasia, mildly elevated and thickened superior cerebellar peduncles (arrowhead), and superior cerebellar dysplasia (arrow) in H1-3. (d–i) Classic molar tooth sign in H2-4 and H3-3. (j–l) Classic molar tooth sign with more severe vermis hypoplasia, cervicomedullary heterotopia (white arrow), and foramen magnum cephalocele (white arrowhead) in H4-3. (a,d,g,j) are T1-weighted images. (b,c,e,f,h,i,k,l) are T2-weighted images.
Supplementary Figure 4 Ciliogenesis rescue experiments with PIBF1 and C21orf2 missense mutations.
Bar graphs show % ciliated mIMCD3 cells following reverse transfection with either scrambled (scr.) negative control or gene-specific pooled siRNAs, and the indicated FLAG-tagged expression constructs. Ciliogenesis was assayed by automated high content imaging in 96-well plates (see On-line Methods and Supplementary Note for details), with additional immunostaining with anti-FLAG for normalization of protein expression levels. Values are derived from six fields of view for n = 3 independent experiments, expressed as percentages of the % ciliated cells value following scrambled siRNA and empty vector transfection (indicated by #), with the statistical significance of pair-wise comparisons as follows: ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001; n.s., not significant (unpaired two-tailed Student’s t-test). Other pair-wise comparisons are indicated by braces. Error bars indicate standard error of the mean.
Supplementary Figure 5 Mutations in C21orf2 cause Jeune syndrome.
(a–f) Overview of Jeune family pedigrees that carry C21orf2 (NM_004928.2) mutations and show segregation consistent with an autosomal recessive inheritance pattern. All affected individuals have cone-rod dystrophy (CRD) with the exception of GC4693.1 shown in (b) who has mild retinal degeneration (RD) marked by an asterisk. The key indicates phenotypic variability of the skeletal features ranging from severe (black symbol), mild (mixed symbol) to absent (grey symbol). Consanguineous unions are indicated by double lines. (e) Electropherogram showing the effect of the c.545 + 1C > T mutation by RT-PCR performed on cDNA derived from RNA isolated from peripheral blood cells from individual F2.1 (lower electropherogram) compared to control individual (upper electropherogram). c.545 + 1C > T results in deletion of five nucleotides at the 3′ end of C21orf2 exon 5, causing a predicted frame-shift of the protein product. (f) Predicted effects of the C21orf2 c.96 + 6 T > A variant on splicing in patient UCL-78.1. Left panel: visualisation of splicing probabilities for wild-type and mutant C21orf2 c.96 + 6 T > A sequence (red text) using Alamut software. Gene Splicer predicts a lack of splicing for the mutant compared to wild-type (wildtype score 1.73, mutant 0; range 0–15); MaxEnt predicts a reduction of splicing probability of 40.1% (reduction from 9.11 to 5.46; range 0–12); NNS splice predicts a reduction of splicing probability for the mutant of 11.6% (wild-type score 0.99, mutant 0.87, range 0–1); SSF predicts a splicing probability reduction for the mutant of 6.3% (wild-type score 88.33, mutant 79; range 0–100) and HSF predicts a splicing probability reduction of 2.7% (wild-type score 87.37, mutant 85.04, range 0–100). Right panel: visualisation of predicted splicing enhancer binding sites using Alamut software shows loss of an SRp55 enhancer (highlighted in yellow) in the mutant C21orf2 c.96 + 6 T > A sequence (red text) compared to wild-type.
Supplementary Figure 6 Subcellular localisation of C21orf2.
(a) Indirect immunofluorescence showing co-localization of C21orf2 (green) with γ-tubulin (red) in ciliary regions in longitudinal sections of adult mouse retinas. Magnified inset indicated by white frame. Abbreviations: CR, ciliary regions of photoreceptor cells; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bar = 50 μm. (b) Immunohistochemical detection of C21orf2 in bone primordia of mouse embryonic ribs and hip joints at E14.5 and E18.5, respectively (arrowheads). Scale bars = 0.1 mm. (c) Upper panel: over-expressed eCFP-C21orf2 (cyan) localizes to the cytoplasm with enrichment at the base of the cilium in hTERT-RPE1 cells (arrowhead). Second panel: eCFP-C21orf2 co-localizes with mRFP-NEK1 (red) in the cytoplasm and at the base of the cilium (arrowhead). Third panel: eCFP-C21orf2 is recruited to the microtubule network via its interaction with mRFP-SPATA7 (red). Lower panel: in the presence of mRFP-SPATA7 (red), both eCFP-C21orf2 (cyan) and 3xFLAG-NEK1 (magenta) are localized to the microtubule network (yellow). Acetylated (Ac) α-tubulin antibody is used as a marker for cilia and the microtubule network (red/magenta/yellow), γ-tubulin antibody is used to stain basal bodies and DAPI (blue) is used to stain nuclei. Primary cilia indicated by arrowheads are shown in magnified insets. Scale bars = 10 μm.
Supplementary Figure 7 Validation of the NEK1 antibody, GST pull-downs of endogenous NEK1 by GST-C21orf2 and GST-SPATA7 fusion proteins from bovine retinal extract, and phenotypes of nek1 morphant zebrafish embryos.
(a) Upper panel: western immunoblot with affinity-purified rabbit polyclonal raised against hNEK1H3T1 (1:2000; red frame) for WCEs from control fibroblasts (C), patient fibroblasts (N) with a homozygous NEK1 nonsense mutation1 in exon 5 treated with either normal media (5) or serum-starved (0), and HEK293 cells (H). Control fibroblasts and HEK293 cells express full-length NEK1 (predicted size 140 kDa) that is absent in patient fibroblasts. Loading control is β-actin (1:3,000; green frame). Lower panel: IF labelling of basal ciliary regions in serum-starved control fibroblasts by affinity-purified anti-NEK1 (green; region in magnified inset indicated by arrowhead) with primary cilia labelled with acetylated (Ac) α-tubulin (red). Scale bar = 10 μm. (b) Upper panel: Simply Blue stain of gel showing 10% input GST negative control (lane 2) and GST-fusion proteins GST-C21orf2 and GST-SPATA7 (lanes 3–4). Lower panel: immunoblot (IB) for endogenous NEK1 on input bovine retinal extract (lane 1) and material pulled down from bovine retinal extract by GST-C21orf2 and GST-SPATA7 (lanes 2–4). (c) Verification of morpholino oligonucleotide (MO) effects using RT-PCR. For the nek1 morpholino oligonucleotide (MO1) targeting the exon6 splice donor site, the wild-type product size is approx. 750 bp whereas MO-injected embryos result in a product of approximately 1250 bp suggesting inclusion of intron 6/7 (intron length 575 bp; see On-line Methods for further details). Amplification of gapdh shows approximately equal cDNA input. (d,e-) H&E staining of eyes of embryos injected with 2ng nek1 MO1 revealed both a reduced outer nuclear layer and generally smaller eyes compared to controls. Abbreviations: INL = inner nuclear layer, ONL = outer nuclear layer. Scale bar = 50 μm. (f,g) 2ng nek1 MO1 injections in rhodopsin:GFP transgenic embryos caused loss of photoreceptor cells (arrows) at 4 dpf compared to controls. Scale bar = 25 μm. (h,i) Acetylated (Ac) α-tubulin staining of nek1 MO1-injected embryos revealed a severe reduction of cilia length in the pronephros compared to control embryos (cilia indicated by arrows). Cilia length appeared normal in the neural tube (data not shown). Scale bar = 10 μm.
Supplementary Figure 8 Full images of SDS-PAGE gels/western blots.
Full unprocessed SDS-PAGE gel images and signals detected on western blotting, with the regions used in the corresponding main display items indicated by red frames.
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Wheway, G., Schmidts, M., Mans, D. et al. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat Cell Biol 17, 1074–1087 (2015). https://doi.org/10.1038/ncb3201
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DOI: https://doi.org/10.1038/ncb3201
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