Unusual dicistronic expression from closely spaced initiation codons in an umbravirus subgenomic RNA

Area of Science:

• Molecular Biology
• Virology
• RNA Biology

Background:

• Translation initiation in RNA viruses often utilizes strategies to maximize coding capacity within limited genomes.
• Pea enation mosaic virus 2 (PEMV2) subgenomic RNA (sgRNA) employs two closely spaced, out-of-frame start codons (AUG26/AUG27) for synthesizing movement/stability proteins.
• Efficient translation from these codons is linked to 3'-proximal cap-independent translation enhancers (3'CITEs).

Purpose of the Study:

• To investigate the mechanism of translation initiation at closely spaced start codons on PEMV2 sgRNA.
• To determine the role of 3'CITEs and sequence context in directing translation of p26 and p27 proteins.
• To elucidate the factors influencing the dicistronic expression from PEMV2 sgRNA.

Main Methods:

• Reporter transcript assays involving modifications of start codon context, inter-codon spacing, and RNA secondary structures.
• Analysis of translation efficiency and initiation rates using polysome profiling.
• Investigating the impact of 5' end modifications on translation.

Main Results:

• Translation initiation at AUG26/AUG27 is 3'CITE-dependent and influenced by sequence context, but not strictly by Kozak rules.
• AUG27 exhibits a repressive effect on upstream AUG26 translation, which is modulated by codon spacing.
• 5' end structure, specifically a hairpin, significantly impacts translation, indicating 5' end dependence of this initiation process.
• 3'CITE-promoted RNA end-to-end interactions affect template number rather than initiation rate.

Conclusions:

• PEMV2 sgRNA utilizes a non-canonical, 3'CITE-driven mechanism for efficient dicistronic expression.
• This mechanism differs from canonical scanning-dependent translation initiation.
• The findings provide insights into viral strategies for maximizing protein production from limited genomic resources.