The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA

Area of Science:

• Molecular Biology
• Yeast Genetics
• Gene Expression Regulation

Background:

• HAC1 mRNA translation in Saccharomyces cerevisiae is repressed by an interaction between its 5'-UTR and intronic sequences.
• This repression is relieved by nonconventional cytosolic splicing of the HAC1 intron, mediated by RNase Ire1, in response to endoplasmic reticulum stress.
• The spliced HAC1 mRNA encodes a transcription factor that enhances cellular protein folding capacity.

Purpose of the Study:

• To elucidate the precise mechanism by which the 5'-UTR and intron interaction (UI) represses HAC1 mRNA translation.
• To identify factors and conditions that lead to the derepression of unspliced HAC1 mRNA translation.

Main Methods:

• Investigated the minimum base-pairing interactions required for UI-mediated repression.
• Utilized overexpression of the helicase eukaryotic initiation factor 4A (eIF4A) to assess its effect on translation.
• Performed genetic screening to identify mutations affecting the UI interaction site.
• Assessed the impact of inserting additional RNA bases between the mRNA 5'-cap and the UI interaction site.

Main Results:

• At least 11 base pairs of interaction between the 5'-UTR and intron are sufficient for translational repression.
• Overexpression of eIF4A derepressed translation of unspliced HAC1 mRNA with 11-bp UI interactions.
• Single mutations within the UI interaction site derepressed translation.
• Adding 24 RNA bases between the 5'-cap and the UI site also led to derepression.

Conclusions:

• The cap-proximal UI-RNA duplex inhibits the recruitment of ribosomes to HAC1 mRNA, causing translational repression.
• Disruption of the UI interaction or facilitating ribosome access can relieve this repression, providing a mechanistic explanation for translational control.