Exogenous RNA surveillance by proton-sensing TRIM25

Exogenous mRNAs require cellular machinery for delivery and translation but also encounter inhibitory factors. To investigate their regulation, we performed genome-wide CRISPR screens with in vitro-transcribed mRNAs in lipid nanoparticles (LNPs). Heparan sulfate proteoglycans and vacuolar ATPase were identified as mediators of LNP uptake and endosomal escape, respectively. TRIM25 emerged as a key suppressor inducing turnover of both linear and circular mRNAs. Endoribonucleases, N4BP1 and KHNYN, along with ZAP, act redundantly in TRIM25-dependent surveillance. TRIM25 specifically targets mRNAs delivered via endosomes, and its RNA affinity increases at acidic pH, suggesting activation by protons released from ruptured endosomes. N1-methylpseudouridine (m1Ψ) modification reduces TRIM25’s RNA binding, helping RNAs evade its suppressive effect. This study comprehensively maps cellular pathways regulating LNP-mRNAs, offering insights into RNA immunity and therapeutics. CRISPR-Cas9 knockout screen on in vitro-transcribed mRNAs with or without N1m-pseudouridine modification delivered by LNP. GFP-stably expressing cell line was included as cellular control.