2-5A treatment of WT and XRN1 KO A549 cells activated RNase L, resulting in detectable and translated mRNA 3' fragments with increased ribosome stalling at cleavage sites.
Fragmented mRNA generated by RNase L causes ribosome stalling, which is modulated by PELO and promotes innate immunity via the ribotoxic stress response.
RNase L is an endonuclease that responds to infections by cleaving most host- and pathogen-derived single-stranded RNAs. This widespread RNA cleavage can lead to death of the infected cell via the ribotoxic stress response (RSR). An ongoing challenge is to understand how RNase L’s endonuclease activity triggers cell death to benefit the host. To address this question, we used nanopore-based long-read sequencing to show that 3’ mRNA fragments in the cell were not fully degraded after RNase L activation and that these fragments were translated by ribosomes. We further asked whether ribosomes on mRNA fragments stall when they reach 3’ ends created by RNase L. We used ribosome profiling to capture footprints protected by these ribosomes, which can be identified by their short length (15-18 nt). We found that RNase L activation increased the number of stalled ribosomes at RNase L cleavage sites. Loss of the ribosome rescue factor PELO increased the number of short footprints derived from stalled ribosomes and augmented the RSR. Our work therefore establishes a role for fragmented mRNA in causing ribosome stalling that promotes innate immunity via the RSR.
Karasik et al. (2026) studied Human lung carcinoma (A549) cells wild type and XRN1 knockout with RNase L activation by 2-5A. 2-5A transfection to activate RNase L vs. Control transfection reagent only (no 2-5A) was evaluated on Detection and characterization of mRNA fragments generated by RNase L cleavage and translation by ribosomes. 2-5A treatment of WT and XRN1 KO A549 cells activated RNase L, resulting in detectable and translated mRNA 3' fragments with increased ribosome stalling at cleavage sites.