Abstract
Cellular responses to changing environments frequently involve rapid reprogramming of the transcriptome. Regulated changes in mRNA degradation rates can accelerate reprogramming by clearing or stabilizing extant transcripts. Here, we measured mRNA stability using 4-thiouracil labeling in the budding yeast Saccharomyces cerevisiae during a nitrogen upshift and found that 78 mRNAs are subject to destabilization. These transcripts include Nitrogen Catabolite Repression (NCR) and carbon metabolism mRNAs, suggesting that mRNA destabilization is a mechanism for targeted reprogramming of the transcriptome. To explore the molecular basis of destabilization we implemented a SortSeq approach to screen the pooled deletion collection library for trans factors that mediate rapid GAP1 mRNA repression. We combined low-input multiplexed Barcode sequencing with branched-DNA single-molecule mRNA FISH and Fluorescence-activated cell sorting (BFF) to identify the Lsm1-7p/Pat1p complex and general mRNA decay machinery as important for GAP1 mRNA clearance. We also find that the decapping modulators EDC3 and SCD6, translation factor eIF4G2, and the 5’ UTR of GAP1 are factors that mediate rapid repression of GAP1 mRNA, suggesting that translational control may impact the post-transcriptional fate of mRNAs in response to environmental changes.
Highlights
Regulated changes in mRNA abundance are a primary cellular response to external stimuli
We investigated changes in mRNA stability using a 4-thiouracil label-chase experiment to track the extant transcriptome during the nitrogen upshift
We found that a functionally enriched set of mRNA are destabilized, including some members of the Nitrogen Catabolite Repression regulon
Summary
Regulated changes in mRNA abundance are a primary cellular response to external stimuli. Changes in mRNA degradation rates have been shown to play a role in responses to heat-shock, osmotic stress, pH increases, and oxidative stress [6, 11,12,13] In response to these diverse stresses destabilization of mRNAs encoding ribosomal-biogenesis gene products, and stress-induced mRNA occurs [6]. Simultaneous increases in both synthesis and degradation rates of some mRNAs may serve to speed the return to a steady-state following a transient pulse of regulation [14]. Destabilization of transcripts can have a delayed effect on reducing protein levels compared to up-regulated genes [18] This suggests that accelerated mRNA degradation may serve additional purposes. Clearance of specific mRNAs could increase nucleotide pools [19] or facilitate reallocation of translational capacity [20,21,22]
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