5′LysTTT tRNA fragments support survival of botulinum-intoxicated neurons by blocking ferroptosis

Botulinum neurotoxins (BoNTs) block cholinergic signaling at neuromuscular junctions, inducing transient muscle paralysis while avoiding neuronal death. However, the mechanism(s) underlying these dual features are yet unknown. Here, we report accumulation of 5'Lys transfer RNA fragments (tRFs) in both BoNT/A-intoxicated cultured human neuroblastoma cells and submandibular glands from BoNT/A-intoxicated rodents. Importantly, we show that 5′LysTTT tRFs balance ferroptosis by cointeracting with the RNA-binding ferroptosis-inducing protein HNRNPM and the 3' untranslated region of the ferroptosis-inhibiting CHAC1 mRNA. Moreover, approximately 20% of the BoNT/A-induced tRFs shared an 11-nucleotide-long LysTTT and LysCTT tRFs-included motif, CCGGATAGCTC, which may target transcripts containing complementary sequences, including the UNC5B transcript that can regulate cell survival. Collectively, the multiple regulatory roles of tRF-5′LysTTT and the shared repetitive motif reveal mechanism(s) supporting the survival of cholinergic neurons under BoNT/A exposure. This understanding may predict the development of novel BoNT/A therapeutic avenues for treating diverse neuromuscular disorders and BoNT/A cosmetic procedures.