Stalled translation on transcripts cleaved by RNase L activates signaling important for innate immunity

Abstract

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 Ls 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/658914v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f08b5forg.highwire.dtl.DTLVardef@14e2cd1org.highwire.dtl.DTLVardef@160814corg.highwire.dtl.DTLVardef@c8ddb4_HPS_FORMAT_FIGEXP M_FIG Stalled translation of mRNAs that are fragmented by RNase L leads to ribosome stalling and potentially collisions. Stalled ribosomes are rescued by PELO or activate innate immune signaling via ZAK. Renderings based on PDB 4o1o and 3jag. C_FIG HighlightsO_LIActivation of RNase L leads to accumulation and translation of mRNA fragments C_LIO_LIRibosomes stall at the 3 end of the RNase L cleaved mRNA fragments C_LIO_LIPELO rescues ribosomes stalled due to RNase L activation C_LI