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

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 three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise 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/CEP90 and C21orf2/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.

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis

“…3D spheroids transfected with siRNAs to deplete cellular levels of any of these four ion channels manifested significantly lower cilia frequency compared to nontargeting siRNA-treated control cells. The reduction in the cilia levels was comparable to that achieved with Ift88 siRNA knockdown, a well-accepted regulator of ciliogenesis used here as a positive control (Pazour et al, 2000;Wheway et al, 2015) (Fig. 2B).…”

“…Non-targeting siRNAs and siRNA targeting human MLNR (which does not target any mouse gene) were used as negative controls. Using this system, cilia are observed as discrete dots above the nuclei (Wheway et al, 2015). The dots were recognized using the Perkin-Elmer 'find spots' algorithm during image analysis.…”

“…The effect of siRNA knockdown on ciliogenesis was assessed by calculating the percentage of cells with a single cilium, with the statistical significance of this effect assessed by calculating z scores (z cilia ). z scores were also calculated for the effect of the knockdown on cell number (z cell ), to exclude effects of cell proliferation or apoptosis on ciliogenesis, because these could be due to non-specific secondary processes (Wheway et al, 2015) (Fig. 1).…”

“…The reverse genetics visual screen was performed as described previously (Wheway et al, 2015). In brief, pools of four Dharmacon siGENOME small interfering RNAs (siRNAs; final total concentration 50 nM) were used to silence gene expression of 340 mouse ion channel genes in mIMCD3 cells (Table S1).…”

C21orf2is mutated in recessive early-onset retinal dystrophy with macular staphyloma and encodes a protein that localises to the photoreceptor primary cilium

“…We suggest these extraocular findings may represent additional ciliopathy manifestations of her C21orf2 mutations. This is further supported by a just-released report in which patients with Jeune syndrome were found to harbour biallelic missense C21orf2 mutations;17 that study combined results from whole-exome sequencing with those from an siRNA-based functional genomics screen for the identification of regulators of ciliogenesis. Some patients in that report had only mild signs of Jeune syndrome, supporting a phenotype continuum of allelic C21orf2 -associated disorders from non-syndromic retinal dystrophy at the mild end and Jeune syndrome at the severe end of the spectrum.…”