A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA

Area of Science:

• Immunology
• Molecular Biology
• Virology

Background:

• Cytosolic helicase retinoic acid-inducible gene-I (RIG-I) detects viral 5'-triphosphorylated RNA (pppRNA) to initiate immune responses.
• Endogenous mRNA is also 5'-triphosphorylated but typically avoids RIG-I activation due to modifications like the 5'-ppp-linked methylguanosine ((m7)G) cap.

Purpose of the Study:

• To investigate the role of methylation status in endogenous mRNA in preventing RIG-I activation.
• To identify the molecular mechanisms by which RIG-I distinguishes between self and non-self RNA.

Main Methods:

• Site-directed mutagenesis of RIG-I to alter specific amino acids in the RNA binding pocket.
• RNA binding assays to assess the interaction of RIG-I with modified and unmodified RNA.
• Knockdown experiments to deplete endogenous methyltransferases.
• Studies utilizing viral methyltransferases.

Main Results:

• The methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) is crucial for preventing RIG-I activation.
• A single amino acid, H830, in the RIG-I RNA binding pocket mediates the steric exclusion of N1-2'O-methylated RNA.
• Mutation of H830 to alanine (RIG-I(H830A)) restored RIG-I binding to N1-2'O-methylated pppRNA, leading to activation by endogenous mRNA.
• Knockdown of endogenous N1-2'O-methyltransferase significantly stimulated RIG-I.
• Viruses, such as yellow fever virus, utilize their 2'O-methyltransferase to evade RIG-I detection.

Conclusions:

• Cap N1-2'O-methylation plays a critical role in RIG-I-mediated tolerance of self-RNA.
• RIG-I's H830 residue is essential for discriminating against N1-2'O-methylated RNA.
• Viruses have evolved mechanisms to exploit this self-RNA recognition pathway to escape innate immunity.