Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy

DDX11 is a DNA helicase involved in critical cellular functions, including DNA replication/repair/recombination as well as sister chromatid cohesion establishment. Bi-allelic mutations of <i>DDX11</i> lead to Warsaw breakage syndrome (WABS), a rare genome instability disorder marked by significant prenatal and postnatal growth restriction, microcephaly, intellectual disability, and sensorineural hearing loss. The molecular mechanisms underlying WABS remain largely unclear. In this study, we uncover a novel role of DDX11 in regulating the macroautophagic/autophagic pathway. Specifically, we demonstrate that knockout of <i>DDX11</i> in RPE-1 cells hinders the progression of autophagy. DDX11 depletion significantly reduces the conversion of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), suggesting a defect in autophagosome biogenesis. This is supported by imaging analysis with a LC3 reporter fused in tandem with the red and green fluorescent proteins (mRFP-GFP-LC3), which reveals fewer autophagosomes and autolysosomes in <i>DDX11</i>-knockout cells. Moreover, the defect in autophagosome biogenesis, observed in DDX11-depleted cells, is linked to an upstream impairment of the ATG16L1-precursor trafficking and maturation, a step critical to achieve the LC3 lipidation. Consistent with this, DDX11-lacking cells exhibit a diminished capacity to clear aggregates of a mutant HTT (huntingtin) N-terminal fragment fused to the green fluorescent protein (HTTQ74-GFP), an autophagy substrate. Finally, we demonstrate the occurrence of a functional interplay between DDX11 and SQSTM1, an autophagy cargo receptor protein, in supporting LC3 modification during autophagosome biogenesis. Our findings highlight a novel unprecedented function of DDX11 in the autophagy process with important implications for our understanding of WABS etiology.<b>Abbreviations</b>: <i>ATG</i> autophagy related; BAF A₁ bafilomycin A₁; CTRL control; DDX11 DEAD/H-box; helicase 11; HF1 healthy donor fibroblasts; HTT huntingtin; KO knockout; MAP1LC3/LC3; microtubule associated protein 1 light chain 3; PLA proximity ligation assay; RPE-1 retinal; pigment epithelial cell line 1; TUBA α-TUBULIN; UBA ubiquitin binding domain; WABS Warsaw breakage syndrome.