NEW YORK (GenomeWeb) – A team of European and North American researchers this week reported the results of a whole-genome siRNA-based reverse genetics screen designed to provide insights into the processes underlying cilia formation, uncovering a number of genetic mutations involved in inherited developmental conditions caused by defects in these organelles.
The primary cilium is a microtubule-based organelle located on the apical surface of most vertebrate cells. These organelles help regulate signaling pathways, including ones of particular importance during embryonic development and patterning in the developing neural tube. Defects in primary cilia are linked with conditions known as ciliopathies that often feature cystic kidney disease, as well as disorders of the central nervous system, eye, and skeleton.
Given that the importance of the primary cilium has only become known in recent years, the mechanisms behind its formation and maintenance are poorly characterized, the investigators wrote in a paper appearing in Nature Cell Biology.
To address this, the team conducted a high-throughput cilia characterization assay to study the involvement of every gene in the formation of the primary cilium.
This, they wrote, "represents a global, hypothesis-neutral approach to identify genes involved in ciliogenesis. It also provides a complementary approach to traditional linkage analysis, candidate gene, or whole-exome sequencing approaches for gene discovery."
While other groups have performed several cell-based medium- or small-scale screens of ciliogenesis on a subset of target genes and uncharacterized components of the cilia proteome, this latest effort represents a "comprehensive view of critical factors involved in ciliogenesis," they noted. Further, it provides a number of candidate gene targets for treatment of ciliopathies.
Specifically, the scientists discovered 112 candidate genes involved in ciliogenesis and ciliopathy, 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.
All three, they wrote, localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells had ciliary defects.
When the group combined the results of the screen with exome sequencing data, it identified recessive mutations in three genes as causes of the ciliopathies Joubert and Jeune syndromes. Using affinity proteomics and co-immunoprecipitation assays, the group was able to place one of these genes, C21orf2, within key ciliopathy-associated protein modules, potentially explaining the skeletal and retinal involvement seen in individuals with C21orf2 variants.
Overall, the study points to the potential of combining different systems biology approaches — in this case, high-content functional genomic screening, whole-exome sequencing, and affinity proteomics, the team stated.
"Our approach has identified ciliary roles for well-studied proteins, identified disease-causing genes, allowed the refinement of patient phenotypes, and highlighted potential disease pathways that could provide deeper insights into cilium biology," the researchers concluded.