SLAM-seq estimates of mRNA half-life changes in S. cerevisiae strains depleted for Dcp2 and Ccr4/Pop2
In eukaryotes, mRNA decay is thought to occur in an ordered manner, with an initial deadenylation step that triggers decapping and RNA degradation. We performed SLAM-seq experiments in yeast strains depleted either for the decapping or for the major deadenylase enzymes to evaluate changes in the half-life of mRNA. Our results suggest that deadenylation and decapping of mRNA are not necessarily linked for mRNA degradation, since the effects of inhibiting the enzymes on RNA stability were not correlated. Overall design: We performed SLAM-seq to analyze changes in mRNA decay after Pop2/Ccr4 depletion, or dcp2 depletion. In short, we grew the cells in SCD media containing low uracil for two doubling times. To pulse label mRNA, we added 4TU to a final concentration of 1mM for 60 min. To deplete the targeted proteins, both Auxin and Estradiol (or only Ethanol, for the undepleted controll) were added to the media (in the presence of 4TU) and the cells were incubated for 60 min. To chase, the cells were filtered and resuspended in SCD containing excess Uracil (20 mM) and no 4TU, in the presence of Auxin and Estradiol, or Ethanol. Samples were collected every 5 mins, followed by RNA-extraction, RNA alkylation with Iodoacetamide, and library preparation using Quantseq for 3'end sequencing. The experiment was performed in two replicates.