A Ψ-Ψ codon-anticodon pairing in nonsense suppression and translational recoding

Area of Science:

• Molecular Biology
• RNA Biology
• Genetics

Background:

• Pseudouridine (Ψ) is a modified uridine with unclear base-pairing flexibility.
• Premature termination codons (PTCs) disrupt protein synthesis, leading to non-functional proteins.

Purpose of the Study:

• To engineer artificial guide RNAs for targeted pseudouridylation of PTCs in mRNA and tRNA.
• To investigate the impact of pseudouridylation on codon-anticodon recognition and PTC readthrough.

Main Methods:

• Engineering artificial box H/ACA guide RNAs for targeted pseudouridylation.
• Utilizing yeast and human cell systems to assess mRNA and tRNA modifications.
• Employing melting curve analysis to determine the affinity of the modified base pair.

Main Results:

• Targeted pseudouridylation created a novel Ψ-Ψ codon-anticodon pair.
• The Ψ-Ψ pair significantly enhanced codon-anticodon recognition and PTC readthrough.
• mRNA levels, tRNA stability, and tRNA charging remained unaffected by pseudouridylation.

Conclusions:

• An unusual Ψ-Ψ base pair was identified, enhancing codon-anticodon recognition.
• This modification robustly promotes PTC readthrough and translational recoding.
• The findings offer a new strategy for modulating protein synthesis via RNA modification.