ALKB-1-dependent tRNA methylation is required for efficient paternal mitochondrial elimination.

Maternal mitochondrial inheritance is secured by mechanisms that exclude paternal mitochondrial DNA (mtDNA). While, epigenetic modifications are vital for spermatogenesis and embryo development, their roles in the paternal mitochondrial elimination (PME) remain poorly understood. Here, we identify ALKB-1, a DNA/RNA demethylase, as a pivotal factor for efficient PME in Caenorhabditis elegans (C. elegans), acting through ALKB-1-dependent modulation of tRNA m(1)A methylation. Mechanistically, ALKB-1 inactivation leads to m(1)A hypermethylation of tRNA, which subsequently disrupts protein translation, impairs mitochondrial proteostasis, and increases ROS levels. This cascade activates the oxidative stress response factor SKN-1/Nrf2 and initiates the mitochondrial unfolded protein response (UPR(mt)) through ATFS-1, causing accumulation of mitochondria and mtDNA in sperm, which ultimately impedes efficient paternal mitochondrial removal and negatively impacts male fertility and embryonic development. Our findings describe a mechanism whereby ALKB-1-mediated tRNA m(1)A epitranscriptomic modifications are necessary for maintaining mitochondrial quality control, thereby influencing PME efficiency, underscoring the importance of this epitranscriptomic stress checkpoint in upholding proper mitochondrial inheritance during reproduction.