In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5′→3′ exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2′-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5′- and 2′-phosphates at its 5′-end that inhibit 5′→3′ decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.
- Jay R Hesselberth
- Patrick D Cherry
- Patrick D Cherry
- Sally E Peach
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Timothy W Nilsen, Case Western Reserve University, United States
© 2019, Cherry et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in E. coli. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3'-UTRs. We also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in E. coli. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in E. coli.
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