Exogenous RNA surveillance by proton-sensing TRIM25

Exogenous messenger RNAs (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 (HSPGs) and vacuolar adenosine triphosphatase (V-ATPase) were identified as mediators of LNP uptake and endosomal escape, respectively. TRIM25-an RNA binding E3 ubiquitin ligase-emerged as a key suppressor inducing turnover of both linear and circular mRNAs. The endoribonucleases N4BP1 and KHNYN, along with the antiviral protein ZAP, act redundantly in TRIM25-dependent surveillance. TRIM25 specifically targets mRNAs delivered by endosomes, and its RNA affinity increases at acidic pH, suggesting activation by protons released from ruptured endosomes. <i>N</i>¹-methylpseudouridine 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.