Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway. ¹ Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors ( e.g. , DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA) ² and traps mutated-DNA-PKcs on ribosomal RNA in vivo, ^3,4 the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive. Intriguingly, while dispensable for murine development, ^5,6 Ku is essential in human cells. ⁷ Despite similar genome sizes, human cells express ∼100-fold more Ku than mouse cells, implying functions beyond NHEJ, possibly through a dose-sensitive interaction with dsRNA, which is ∼100 times weaker than with dsDNA. ^2,8 While investigating the essentiality of Ku in human cells, we found that depletion of Ku - unlike LIG4 - induces profound interferon (IFN) and NF-kB responses reliant on the dsRNA-sensor MDA5/RIG-I and adaptor MAVS. Prolonged Ku-degradation also activates other dsRNA-sensors, e.g. PKR that suppresses protein translation, and OAS/RNaseL that cleaves rRNAs and eventually induces growth arrest and cell death. MAVS, RIG-I, or MDA5 knockouts suppressed IFN signaling and, together with PKR knockouts, partially rescued Ku-depleted human cells. Ku-irCLIP analyses revealed that Ku binds to diverse dsRNA, predominantly stem-loops in primate-specific Alu elements ⁹ at anti-sense orientation in introns and 3’-UTRs. Ku expression rose sharply in higher primates tightly correlating with Alu-expansion (r = 0.94/0.95). Together, our study identified a vital role of Ku in accommodating Alu-expansion in primates by mitigating a dsRNA-induced innate immune response, explaining the rise of Ku levels and its essentiality in human cells.