These authors contributed equally to this work.
A Homozygous PDE6D Mutation in Joubert Syndrome Impairs Targeting of Farnesylated INPP5E Protein to the Primary Cilium
Version of Record online: 13 NOV 2013
© 2013 WILEY PERIODICALS, INC.
Volume 35, Issue 1, pages 137–146, January 2014
How to Cite
Thomas, S., Wright, K. J., Corre, S. L., Micalizzi, A., Romani, M., Abhyankar, A., Saada, J., Perrault, I., Amiel, J., Litzler, J., Filhol, E., Elkhartoufi, N., Kwong, M., Casanova, J.-L., Boddaert, N., Baehr, W., Lyonnet, S., Munnich, A., Burglen, L., Chassaing, N., Encha-Ravazi, F., Vekemans, M., Gleeson, J. G., Valente, E. M., Jackson, P. K., Drummond, I. A., Saunier, S. and Attié-Bitach, T. (2014), A Homozygous PDE6D Mutation in Joubert Syndrome Impairs Targeting of Farnesylated INPP5E Protein to the Primary Cilium. Hum. Mutat., 35: 137–146. doi: 10.1002/humu.22470
Contract grant sponsors: Agence Nationale de la Recherche (ANR 1122 01 FOETOCILPATH); IMAGINE Institute; Fondation pour la Recherche Médicale (FRM DEQ20071210558); National Institutes of Health (DK053093, DK070263, NS048453); European Research Council (ERC Starting Grant 260888); The Italian Ministry of Health (Ricerca Corrente 2012 and Ricerca Finalizzata Malattie Rare 2008).
Communicated by Ming Qi
- Issue online: 18 DEC 2013
- Version of Record online: 13 NOV 2013
- Accepted manuscript online: 25 OCT 2013 11:43AM EST
- Manuscript Accepted: 10 OCT 2013
- Manuscript Received: 30 JAN 2013
- Agence Nationale de la Recherche. Grant Number: ANR 1122 01 FOETOCILPATH
- Fondation pour la Recherche Médicale. Grant Number: FRM DEQ20071210558
- National Institutes of Health. Grant Numbers: DK053093, DK070263, NS048453
- European Research Council (ERC Starting). Grant Number: 260888
- The Italian Ministry of Health
Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.
Figure S1. Exome sequencing. Exome capture with the SureSelect Human All Exon kit (Agilent) was followed by single-end sequencing on an Illumina Genome Analyzer. (a) Filtering of the data identified a unique truncating mutation within the targeted homozygous region. (b) Polyphen 2 analysis of the RAB17 R183K predicts it is benign. Arg at position 183 is not conserved and is replaced by a lysin in Dog (Canis familiaris) and Duckbill platypus (Ornithorhynchus anatinus).
Figure S2. PDE6D expression pattern during human development. Immunohistochemistry was performed on normal human embryos obtained after elective pregnancy termination in agreement with French legislation (law no. 2004-800), National Ethics Committee recommendations (report no. 1 of May 22, 1984) and the Necker Hospital ethics committee. Embryonic stages were established according to Carnegie staging (CS) classification. After citrate buffer antigen retrieval treatment, adjacent sections were incubated overnight with or without rabbit polyclonal anti-PDE6D antibody (Sigma, HPA037434) and subsequently with goat polyclonal anti-rabbit IgG coupled to alkaline phosphatase (AbCam, ab6722).
PDE6D is ubiquitously expressed within human embryonic tissues with a more intense staining in the entire central nervous system (prosencephalon (Pr), mesencephalon (Mes) and rhombencephalon (Rh) (a,b), in renal tubules (c) and in epithelial cells of the bronchus (d). PDE6D is also localized in the limb bud mesenchyme (e) and in the neural layer of the optic cup (future neural retina) (f). Br, bronchus; Cb, cerebellum; H, heart; L, lens; nR, neural retina; pR, pigment layer of retina; R, Rathke's pouch; sc, spinal cord; tg, tongue; Tu, renal tubules.
Figure S3. Zebrafish pde6d morpholino knockdown. (a) Control zebrafish larva at 3 dpf. (b) pde6d morphant at 3 dpf after injection of an exon 2 splice donor-blocking morpholino oligonucleotide (MO), (Gene-Tools, LLC) with sequence 5’- TAATGCTGAGAACCACAAACCTTCA-3’, or a translation blocking MO 5’- TTCGTCCGAAGACATCTTTTTCCTT-3’. pde6d-deficient embryos exhibit defects in eye development and pericardial edema (arrowhead). A control random sequence morpholino did not cause a phenotype at similar injection concentration. (c) RTPCR analysis of control and morphant mRNA revealed near complete absence of pde6d mRNA in exon 2 splice donor morphants (MO) compared with controls (wt).
Figure S4. Isolation of PDE6D interacting proteins. (A) Silver stained gel showing the eluate from tandem affinity purification of LAP-tagged PDE6D. (B) Table of proteins with or without a CaaX-box identified by mass spectrometry as PDE6D interactors. Asterisk denotes a CaaX-box predicted to be geranylgeranylated.
Figure S5. Partial co-localization of PDE6D and INPP5E. GFP-tagged PDE6D localizes to the transition zone and proximal end of the cilium while INPP5E localizes uniformly along the length of the axoneme.
Figure S6. PDE6D expression is required for INPP5E localization to the primary cilium. (a) Efficiency of PDE6D siRNAs tested by quantitative RT-PCR performed on RNA extracted from RPE cells 48 hours after transfection with either control or two different siRNA oligos targeting PDE6D (Dharmacon, J-004310-09 and J-004310-11). (b) Immunocytochemistry using antibodies raised against INPP5E, pericentrin and acetylated alpha tubulin (α-ac-Tub) in RPE1 cells transfected with either a control siRNA or a siRNA targeting PDE6D and showing that PDE6D extinction leads to to a near total loss of INPP5E localization to the primary cilium as quantified in the graph. Scale bars are 10 um. Graph is average of three independent experiments plus standard deviation.
Figure S7. Western blot of INPP5E on proteins extracted from control and patient fibroblasts. INPP5E protein is expressed in both patient and control fibroblasts. Antibodies used: INPP5E (Proteintech, 17797-1-AP) and beta-actin (Santa Cruz Biotechnology; sc-81178).
Figure S8. INPP5E primary cilia localization is independent from ARL2, ARL3 and RP2. (A) GST pulldowns were performed using GST-ARL2/3 TN (GDP mimicking ARL2/3 mutant protein) or GST-ARL2/3 QL (GTP mimicking ARL2/3 mutant protein) and in vitro translated myc-tagged WT or Δexon3-PDE6D. WT PDE6D binds to ARL2/3 in a GTP dependent manner while Δexon3-PDE6D is unable to bind either ARL2 or ARL3. (B) RPE cells were transfected with LAP6-PDE6D and myc-INPP5E, then lysed and incubated with increasing amount of either GDP (TN)- or GTP (QL)-mimicking mutant GST-ARL2 or GST-ARL3 as indicated. Immunoprecipitation of PDE6D with anti-GFP antibodies and detection of co-immunoprecipitated INPP5E by anti-Myc immunoblotting reveal specific ARL3 QL-dose dependent interaction between INPP5E and PDE6D. These results indicate that GTP-ARL3 but not GTP-ARL2-binding efficiently releases INPP5E from PDE6D. (C) Immunocytochemistry using antibodies raised against INPP5E, pericentrin and acetylated alpha tubulin (α-ac-Tub) in RPE1 cells transfected with either control siRNA or siRNA directed against ARL2, ARL3, and RP2 as previously described (Wright K.J. et al. 2011) and showing that INPP5E ciliary localization is not perturbed by ARL2, ARL3 or RP2 extinction. Scale bars are 10 μm. Graph is average of three independent experiments plus standard deviation.
Table S1. Primers used for PDE6D genomic sequencing, RT-PCR and mutagenesis
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