Intraflagellar transport protein IFT172 contains a C-terminal ubiquitin-binding U-box-like domain involved in ciliary signaling

Intraflagellar transport (IFT) is a fundamental process driving ciliogenesis in most eukaryotic organisms. IFT172, the largest protein of the IFT complex, plays a crucial role in cilium formation and is associated with several disease variants causing ciliopathies. While IFT172 is tethered to the IFT-B complex via its N-terminal domains, the function of its C-terminal domains has remained elusive. Here, we reveal that the C-terminal part of IFT172 interacts with IFT-A complex subunits, providing a molecular basis for the role of IFT172 in bridging IFT-A and IFT-B complexes. We determine the crystal structure of the C-terminal part of IFT172, uncovering a conserved U-box-like domain often found in E3 ubiquitin ligases. This domain exhibits ubiquitin-binding properties and auto-ubiquitination activity. The IFT172 auto-ubiquitination activity is reduced in the C1727R patient ciliopathy variant. We use CRISPR-engineered RPE-1 cells to demonstrate that the U-box-like domain is essential for IFT172 protein stability and proper cilium formation. Notably, RPE-1 cells with heterozygous deletion of the U-box domain show altered TGFB signaling responses, particularly in SMAD2 phosphorylation levels and AKT activation. Our findings suggest a novel dual role for IFT172 in both structural support within IFT trains and regulation of ciliary ubiquitination and signaling pathways, providing new insights into the molecular mechanisms underlying IFT172-related ciliopathies.