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Abstract
Translation of HAC1 mRNA in the budding yeast Saccharomyces cerevisiae is derepressed when RNase Ire1 removes its intron via nonconventional cytosolic splicing in response to accumulation of unfolded proteins inside the endoplasmic reticulum. The spliced HAC1 mRNA is translated into a transcription factor that changes the cellular gene expression patterns to increase the protein folding capacity of cells. Previously, we showed that a segment of the intronic sequence interacts with the 5′-UTR of the unspliced mRNA, resulting in repression of HAC1 translation at the initiation stage. However, the exact mechanism of translational derepression is not clear. Here, we show that at least 11-base-pairing interactions between the 5′-UTR and intron (UI) are sufficient to repress HAC1 translation. We also show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 5′-UTR and intronic sequences. In addition, our genetic screen identifies that single mutations in the UI interaction site could derepress translation of the unspliced HAC1 mRNA. Furthermore, we show that the addition of 24 RNA bases between the mRNA 5′-cap and the UI interaction site derepressed translation of the unspliced HAC1 mRNA. Together, our data provide a mechanistic explanation for why the cap-proximal UI–RNA duplex inhibits the recruitment of translating ribosomes to HAC1 mRNA, thus keeping mRNA translationally repressed.
Highlights
The transacting regulatory protein(s) or noncoding RNA(s) [3]
Under stress conditions, such as when unfolded proteins overaccumulate inside the endoplasmic reticulum (ER), HAC1 mRNA colocalizes with an ER-resident RNase Ire1 that cleaves out the inhibitory intron [9, 10]. tRNA ligase ligates two cleaved exons [11], generating a matured mRNA that yields Hac1 protein [12, 13]
We show that three RNA bases (C-23, C-27, and C-32) at the UI interaction site play a major role in the translational regulation of HAC1 mRNA
Summary
The cap-proximal RNA secondary structure inhibits preinitiation complex formation on HAC1 mRNA. We show that overexpression of the helicase eukaryotic initiation factor 4A derepressed translation of an unspliced HAC1 mRNA containing only 11-bp interactions between the 50-UTR and intronic sequences. We and others have shown that the 50-UTR–intron (UI)–RNA duplex inhibits the initiation of HAC1 translation [6, 8] Under stress conditions, such as when unfolded proteins overaccumulate inside the endoplasmic reticulum (ER), HAC1 mRNA colocalizes with an ER-resident RNase Ire that cleaves out the inhibitory intron [9, 10]. Under certain conditions, cap-dependent translation initiation occurs without ribosomal scanning [17] It is not yet clear how and to what extent the UI–RNA duplex in HAC1 mRNA affects the assembly of ribosomes and other factors to form the PIC or its movement along the 50-UTR.
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