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

Area of Science:

• Molecular Biology
• Immunology
• Cell Biology

Background:

• Ribonuclease L (RNase L) is an endonuclease crucial for the innate immune response, cleaving single-stranded RNAs during infection.
• RNase L activation triggers the ribotoxic stress response (RSR), a pathway leading to infected cell death.
• The precise mechanism by which RNase L-mediated RNA cleavage induces cell death and benefits the host remains incompletely understood.

Purpose of the Study:

• To investigate how RNase L's endonuclease activity triggers cell death.
• To determine if fragmented messenger RNA (mRNA) plays a role in the ribotoxic stress response (RSR).
• To explore the impact of ribosome stalling on mRNA fragments generated by RNase L.

Main Methods:

• Nanopore-based long-read sequencing to analyze mRNA fragments after RNase L activation.
• Ribosome profiling to identify and quantify ribosome footprints on fragmented mRNA.
• Analysis of the ribosome rescue factor PELO's role in the RSR.

Main Results:

• 3' mRNA fragments generated by RNase L were found to be translated by ribosomes.
• RNase L activation led to increased ribosome stalling at RNase L cleavage sites on mRNA fragments.
• Loss of the PELO factor exacerbated ribosome stalling and augmented the RSR.

Conclusions:

• Fragmented mRNA resulting from RNase L activity causes ribosome stalling.
• This ribosome stalling on fragmented mRNA promotes innate immunity via the RSR.
• The study establishes a novel mechanism linking RNA fragmentation, ribosome dynamics, and innate immune signaling.