Exogenous RNA surveillance by proton-sensing TRIM25

Area of Science:

• Molecular Biology
• Immunology
• Biotechnology

Background:

• Exogenous messenger RNAs (mRNAs) are crucial for therapeutics but face cellular barriers and regulatory mechanisms.
• Understanding the cellular pathways governing the fate of externally delivered mRNAs is essential for optimizing their efficacy.

Purpose of the Study:

• To comprehensively map the cellular pathways regulating lipid nanoparticle (LNP)-delivered mRNAs.
• To identify key factors involved in mRNA uptake, endosomal escape, and degradation.
• To elucidate the role of RNA-binding proteins and modifications in mRNA regulation and immune response.

Main Methods:

• Genome-wide CRISPR screens were employed using in vitro-transcribed mRNAs encapsulated in LNPs.
• Investigated the roles of heparan sulfate proteoglycans (HSPGs) and vacuolar adenosine triphosphatase (V-ATPase) in LNP delivery.
• Characterized the function of TRIM25, N4BP1, KHNYN, and ZAP in mRNA surveillance and turnover.

Main Results:

• Heparan sulfate proteoglycans (HSPGs) mediate LNP uptake, and V-ATPase facilitates endosomal escape.
• TRIM25, an RNA-binding E3 ubiquitin ligase, acts as a major suppressor of both linear and circular mRNA turnover.
• TRIM25-dependent surveillance involves redundant endoribonucleases (N4BP1, KHNYN) and the antiviral protein ZAP.
• TRIM25 activity is enhanced by acidic endosomal pH and specifically targets endosome-delivered mRNAs.
• The N1-methylpseudouridine modification in mRNA reduces TRIM25 binding, promoting evasion of cellular suppression.

Conclusions:

• This study provides a comprehensive map of cellular pathways regulating LNP-delivered mRNAs.
• Identified TRIM25 as a critical regulator of mRNA stability, with its activity modulated by endosomal environment and mRNA modifications.
• Findings offer crucial insights into RNA immunity and provide a foundation for developing more effective mRNA-based therapeutics.