Abstract

Mitochondrial ribosome biogenesis depends on RNA helicases such as DDX28, a DEAD-box helicase that plays an essential role during early mitoribosome large-subunit assembly by interacting with 16S rRNA. Here, we demonstrate that the helicase core domain of DDX28 binds sequence and structure specifically to the H88_L stem-loop in 16S rRNA, with the RecA2 domain residue M431 as a key determinant for substrate selectivity. The N-terminal disordered region of DDX28 enhances nonspecific RNA binding but does not contribute to enzymatic activity. Furthermore, DDX28 deficiency disrupts mitochondrial translation, impairs OXPHOS complex assembly, and leads to metabolic dysfunction, including reduced membrane potential, elevated ROS, and suppressed glycolysis. Transcriptomic and metabolomic analyses reveal a compensatory upregulation of ribosome biogenesis genes alongside a dysregulation of the TCA cycle, oxidative phosphorylation, and lipid metabolism. Our integrated structural and functional study establishes DDX28 as an essential factor for mitoribosome assembly with potential links to mitochondrial disorders.