P-bodies act as sites for dynamic RNA processing

Processing bodies (PBs) are cytoplasmic granules that function in the cellular response to stress conditions by regulating mRNA metabolism. They are thought to act as sites where non-translating mRNAs are localised to undergo degradation or sequestration, but their exact intracellular roles remain unclear. We used SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) to study PB-localisation and global mRNA fate during glucose depletion conditions that induce PB formation. This enabled us to differentiate newly synthesized and pre-existing RNAs and to separately track mRNA synthesis and degradation. We show that pre-existing mRNAs localise to PBs with differing kinetics with some transcripts localising over the time-course of glucose starvation and some transcripts localising in a more dynamic manner. We identified a small number of transcripts that are transiently enriched in PBs and appear to depend on this localisation for decay. However, most transiently localised transcripts are not destabilized following glucose starvation and PBs appear to act as temporary storage sites for transcripts that later undergo alternative fates. For other transcripts, both their pre-existing and newly made transcripts accumulate in PBs over the time-course of glucose depletion and we suggest that these transcripts are important for adaptation once the nutrient stress is relieved. Together, our data indicate that different classes of mRNAs behave differently following nutrient depletion with P-bodies predominantly acting as triage sites for mRNAs with a more limited role in mRNA-degradation. Overall design: SLAM-seq profiling of total and P-body–associated mRNAs in Saccharomyces cerevisiae W303 cells, with total RNA sampled under glucose-replete and glucose-depletion conditions and P-body–associated RNA sampled under glucose-depletion at 0, 2.5, 5, 10, 20, and 40 minutes of starvation.