DNAAF5

DNAAF5 is a cytoplasmic dynein axonemal assembly factor required for the function of motile cilia (PMID:37104040, PMID:36712068). It promotes the assembly of dynein motor complexes prior to their incorporation into the axoneme, such that its loss reduces both axonemal regulatory and structural proteins and produces severe cilia motor assembly defects; complete loss-of-function is embryonic lethal in mice (PMID:37104040, PMID:36712068). The requirement for DNAAF5 is allele- and tissue-specific: the same missense variant yields divergent cilia function across different multiciliated tissues, indicating tissue-specific molecular requirements for dynein arm assembly (PMID:37104040, PMID:36712068). This role is conserved in vertebrate development, where dnaaf5 is expressed in motile ciliated tissues of zebrafish embryos and its loss produces ciliopathic phenotypes (PMID:42029186). In a non-ciliary context, DNAAF5 acts as a scaffold that directly binds PFKL and recruits the deubiquitinase USP39, forming a ternary complex that stabilizes PFKL by promoting its deubiquitination and thereby enhancing glycolysis in hepatocellular carcinoma cells (PMID:36276075).

1. 2022 Medium

   Identified a non-ciliary, scaffolding function for DNAAF5, establishing that it can bridge a substrate to a deubiquitinase to control protein stability and metabolic output.

   Evidence Co-IP, mass spectrometry, knockout/overexpression cell lines, xenografts, and USP39 knockdown rescue in hepatocellular carcinoma cells

   PMID:36276075

   Open questions at the time

   • Single lab, non-ciliary context may represent a gain-of-function or context-restricted role
   • Structural basis of the DNAAF5–PFKL–USP39 ternary complex not resolved
   • Relationship between this glycolytic scaffold role and the cilia assembly function unknown
2. 2023 High

   Established DNAAF5 as a dynein motor assembly factor essential for motile cilia, defining its in vivo requirement and the proteomic consequences of its loss.

   Evidence CRISPR-Cas9 engineered missense and frameshift-null Dnaaf5 alleles in mice, with cilia ultrastructure, proteomics of isolated airway cilia, and transcriptional readouts

   PMID:36712068 PMID:37104040

   Open questions at the time

   • Precise biochemical step in dynein arm preassembly catalyzed or scaffolded by DNAAF5 not defined
   • Direct dynein-chain or co-chaperone partners not identified
3. 2023 Medium

   Revealed that DNAAF5 dependence is allele- and tissue-specific, explaining how a single variant can produce divergent cilia phenotypes across multiciliated tissues.

   Evidence Comparative ultrastructure and proteomic profiling of cilia from multiple multiciliated tissues in Dnaaf5 missense homozygous and compound heterozygous mice

   PMID:36712068 PMID:37104040

   Open questions at the time

   • Molecular basis of tissue-specific dynein arm assembly requirements not identified
   • Single-lab allelic series
4. 2025 Medium

   Demonstrated evolutionary conservation of the motile cilia role, showing DNAAF5 acts in motile ciliated tissues during vertebrate development.

   Evidence In situ hybridization expression mapping and CRISPR crispant phenotyping in zebrafish embryos

   PMID:42029186

   Open questions at the time

   • Single method per finding; crispant rescue not described
   • Molecular partners in zebrafish not defined

• How DNAAF5 reconciles its cytoplasmic dynein preassembly role with the non-ciliary glycolytic scaffold function, and the precise biochemical step it performs in dynein arm assembly, remain unresolved.
• No structural model of DNAAF5 or its complexes
• Direct dynein assembly substrates/co-factors unidentified
• Whether scaffolding for deubiquitination is a general DNAAF5 mechanism is unknown