Single molecule mRNA decay measurements in eukaryotes reveal unique regulatory mechanisms

Abstract

mRNA turnover is an important determinant of cytoplasmic mRNA levels and measuring it accurately is essential to an understanding as to how it is regulated. We developed a novel approach to quantify changes in mRNA decay rates and used it to evaluate two cyclical genes in minimally perturbed, single yeast cells. We used yeast morphological markers to determine the length in minutes of the cell cycle. Using single molecule FISH we detected both cytoplasmic and nascent transcripts and determined the decay rates using a mathematical model. We found that the stability of two cyclically expressed genes, SWI5 and CLB2, changed depending on the cell cycle and that this regulation was coordinated with, and controlled by their promoter activity. Prior to anaphase onset, inhibition of mRNA decay allowed for transcript buildup and afterwards rapid decay prevented carry-over of G2 phase specific mRNAs into the new cell cycle. This switch in mRNA stability was regulated by the promoter sequence and was independent of specific sequences found in the mRNAs. We identified Dbf2p/20p kinases as regulators of SWI5 and CLB2 decay and found Dbf2p localized at the transcription site of SWI5 and CLB2 genes using chromatin immunoprecipitation (ChIP) suggesting that Dbf2p associates with SWI5 and CLB2 co-transcriptionally. In this model the specific decay machinery is first recruited by the promoter and then co-transcriptionally deposited onto the mRNA. After export, the appropriate cytoplasmic queues initiate the decay process. Thus, promoter-dependent activity in the nucleus directly influences how and when an mRNA will be degraded in the cytoplasm. This novel approach for measuring mRNA decay rates will represent a platform for the analysis of proteins involved in the regulation of cell cycle-dependent mRNA decay.