Eukaryotic tRNA ligases mediate RNA break repair

RNA is continuously exposed to damage during physiological metabolism and stress, yet cellular responses to RNA damage remain less understood than DNA repair pathways. RNA strand breaks are particularly deleterious because they generate chemically incompatible RNA ends. Eukaryotic tRNA ligases have been implicated in RNA processing and repair, but whether they function as general RNA repair enzymes remains unresolved. Here, we show that the evolutionarily divergent tRNA ligases, human RTCB and fungal Trl1, mediate RNA break repair (RBR) targeting ribosomes and other ribonucleoprotein (RNP) complexes. Using direct RNA nanopore sequencing, we map these repair events at nucleotide resolution, demonstrating that tRNA ligases repair breaks in ribosomal RNA and restore translational activity of repaired ribosomes. We further identify repair across additional structured cellular RNAs. We show that loss of RBR activity leads to RNA fragmentation in human cells and impairs cell viability upon oxidative stress. Together, these findings uncover a broader role for eukaryotic tRNA ligases in repairing RNA breaks and maintaining transcriptome integrity.