Ciliopathy-associated mutations of IFT122 impair ciliary protein trafficking but not ciliogenesis

Abstract

The intraflagellar transport (IFT)machinery containing the IFT-A and IFT-B complexesmediates ciliary protein trafficking. Mutations in the genes encoding the six subunits of the IFT-A complex (IFT43, IFT121, IFT122, IFT139, IFT140, and IFT144) are known to cause skeletal ciliopathies, including cranioectodermal dysplasia (CED). As the IFT122 subunit connects the core and peripheral subcomplexes of the IFT-A complex, it is expected to play a pivotal role in the complex. Indeed, we here showed that knockout (KO) of the IFT122 gene in hTERT-RPE1 cells using the CRISPR/Cas9 systemled to a severe ciliogenesis defect, whereas KO of other IFT-A genes hadminor effects on ciliogenesis but impaired ciliary protein trafficking. Exogenous expression of not only wild-type IFT122 but also its CED-associatedmissensemutants, which fail to interact with other IFT-A subunits, rescued the ciliogenesis defect of IFT122-KO cells. However, IFT122-KO cells expressing CED-type IFT122mutants showed defects in ciliary protein trafficking, such as ciliary entry of Smoothened in response to Hedgehog signaling activation. The trafficking defects partially resembled those observed in IFT144-KO cells, which demonstrate failed assembly of the functional IFT-A complex at the base of cilia. These observations make it likely that, although IFT122 is essential for ciliogenesis, CED-type missense mutations underlie a skeletal ciliopathy phenotype by perturbing ciliary protein trafficking withminor effects on ciliogenesis per se.