Cloning and characterization of novel ccdc103 interactors

Abstract

Coiled-coil domain containing protein 103 is a homodimeric protein that functions as a dynein attachment factor in motile cilia. Ccdc103 defects in zebrafish (Danio rerio) embryos lead to paralyzed motile cilia, pronephric cysts, hydrocephalus, and a curved body axis. In humans, CCDC103 mutations cause primary ciliary dyskinesia (PCD), a genetic syndrome characterized by bronchial scarring, chronic respiratory infections, and infertility. Studies are just beginning to uncover the function of Ccdc103 in both zebrafish and humans, but, apart from foxj1a regulation and dynein arm attachment, little else is known about its interactors or its place in cellular pathways. We identified 14 potential interactors through a yeast-two-hybrid study using mouse Ccdc103 as bait against a mouse cDNA prey library. One of the identified interactors is ankyrin and EF-hand containing protein 1 (Ankef1), which has two paralogs (ankef1a and ankef1b) in zebrafish. Both paralogs share the same ankyrin—EF-hand—ankyrin domain structure and display conserved synteny with SNAP25 in zebrafish, frogs, mice, and humans. We first cloned ankef1a and ankef1b, then used whole-mount in situ hybridization (WISH) to identify the embryonic expression patterns of both paralogs. WISH and sectioning showed that ankef1 is ubiquitously expressed during the first 24 hours of zebrafish development, with more restricted expression in the pharynx, swim bladder, and otic vesicles through 5 days post-fertilization (dpf). Quantitative reverse transcriptase PCR (qPCR) was used to quantify ankef1 transcript levels in embryos and adult tissues. qPCR data showed that both transcripts are maternally contributed and increase later in development after an initial drop in expression in the first two days. Ankef1a was also highly expressed in the testis, which is a major site of ANKEF1 expression in humans. We also generated knockout (KO) lines of ankef1a using CRISPR/Cas9 technology. An ankef1b KO line was obtained from ZIRC. Future studies on mutant phenotypes will reveal potential functional overlap between Ankef1a and Ankef1b.