OFD1 Knockout Cell Line (HEK293)

BACKGROUND:Processing bodies (P-bodies) are nonmembranous ribonucleoprotein (RNP) granules located in the cytosol that function as assembly hubs for RNA storage and degradation. Although there are reports indicating that certain P-body proteins are also present at the centrosome and participate in primary cilia development, how these P-body proteins localize to the centrosome remains unclear. In mammalian cells, coiled-coil alpha-helical rod protein 1 (CCHCR1) is localized to both the P-bodies and centrosomes, where it interacts with the P-body component enhancer of mRNA-decapping protein 4 (EDC4) as well as a range of centriolar satellite components, yet its cellular function remains poorly characterized. METHODS:Biotin identification (BioID) coupled with mass spectrometry, immunoprecipitation (IP), glutathione S-transferase (GST) pull-down, and acceptor bleaching fluorescence resonance energy transfer (AB-FRET) assay were used to explore and identify protein-protein interactions. Gene overexpression, RNA interference-based gene knockdown, CRISPR-Cas9-mediated gene knockout, and immunofluorescence (IF) were applied to elucidate the underlying molecular mechanism. RESULTS:We identified that CCHCR1 interacts with oral-facial-digital syndrome 1 protein (OFD1) via its C-terminal coiled-coil domain. The centrosomal localization of CCHCR1 is determined by OFD1 and pericentriolar materials 1 (PCM1). We also found that CCHCR1 recruits P-body proteins to the centrosome through interacting with EDC4 via its N-terminal coiled-coil domain. Depletion of either CCHCR1 or P-body components EDC4 and DEAD-Box Helicase 6 (DDX6) impairs ciliogenesis. CONCLUSIONS:CCHCR1 acts as a linker that recruits P-body proteins to the centrosome and is essential for cilia development. The recruitment of P-body proteins to the centrosome via CCHCR1 is also one of the mechanisms by which PCM1 and OFD1 are involved in ciliogenesis.