Precise analysis of decay kinetics is necessary to understand when and how a decay regulator functions and single-cell, single-molecule techniques could advance our understanding of mRNA turnover. For example, the kinetic behavior of individual RNA polymerase II (RNAPII) transcribing a gene (reviewed in (Ardehali and Lis, 2009
)) provides a precise quantification of the contribution of mRNA synthesis to the cellular pool of transcripts. However, to date no such approach has been available for measuring mRNA turnover. Traditional techniques have relied on normalization of decay signal and on a large sample of cells, genetically modified or treated with inhibitors, to stop transcription and thus obtain kinetic information of a decaying mRNA species (reviewed in (Passos and Parker, 2008
)). Furthermore, the accuracy of decay measurement varies with the technique used. For example, in budding yeast, half-lives of an individual mRNA species quantified by different approaches may differ by more than 50 percent (Grigull et al., 2004
; Holstege et al., 1998
; Wang et al., 2002
). In turn, the accuracy of the decay curve will influence how precisely it can be modeled. In this work we use single-molecule counting with Fluorescent In Situ Hybridization (FISH) (Zenklusen et al., 2008
) to derive an absolute measure of mRNA synthesis and decay in individual cells. This provided a highly sensitive approach for detecting changes occurring in a fraction of cells, that otherwise would have been obscured.
We focused on mRNA turnover since it could regulate gene expression during the cell cycle. For instance, entry into mitosis induces a rapid mRNA decay of the mitotic Clb2p cyclin that, if prevented, can cause failure of cells to finish mitosis (Cai et al., 2002
; Gill et al., 2004
). Entry into G0 causes stabilization of specific G0 mRNAs (Talarek et al., 2010
), whereas the stability of the canonical histone mRNAs increases with the onset of S phase and exit from S phase induces their rapid decay (Marzluff et al., 2008
; Osley, 1991
). Thus, together with their cyclical transcription, the destabilization of mRNAs can restrict the activity of periodically expressed genes to a particular cell cycle phase. This modulation of stability is typically achieved through binding of decay regulators to specific sequences located in the mRNA (reviewed in (Guhaniyogi and Brewer, 2001
We focused on two cell cycle regulated genes, SWI5
and measured changes in their mRNA turnover during the cell cycle. Swi5p is a transcription regulator of late mitosis genes and Clb2p is a G2 phase cyclin that drives the progression of cells towards mitosis. They are co-regulated through shared promoter elements (Koranda et al., 2000
; Spellman et al., 1998
; Zhu et al., 2000
) and were measured to degrade with 8 min and 4.5 min half-lives, respectively (Wang et al., 2002
). We used morphological markers to determine timing of the cell cycle. We counted absolute numbers of cytoplasmic and nascent transcripts (Zenklusen et al., 2008
) and analyzed decay rates using a mathematical model without the use of transcriptional inhibitors, genetic mutants, or the need to normalize mRNA signal.
The use of a single-molecule mRNA decay measurement enabled identification of a novel regulatory pathway of mRNA decay that provides an additional level of cell cycle regulation. We determined that the half-life of SWI5 and CLB2 decreases more than 30 fold with the onset of prometaphase/metaphase. Furthermore, regulation of this mRNA decay is coordinated with their transcription and controlled by their promoter sequence, independent of the specific cis sequences located in the mRNA. By using morphological markers, we were able to determine that the cell cycle progression and the prometaphase/metaphase stability switch of SWI5 and CLB2 were coupled and regulated by the mitotic exit network kinases, Dbf2p and Dbf20p. Both kinases bind to SWI5 and CLB2 mRNAs, while Dbf2p is also enriched at their transcription sites. We propose a model whereby Dbf2p is first recruited by the promoter and then co-transcriptionally deposited onto the mRNA. Once in the cytoplasm, the mRNA associates with Dbf20p, and then waits for the appropriate cellular cues to initiate the decay process. Thus, for a subset of budding yeast mRNAs, promoter dependent activity directly influences how and when an mRNA will be degraded in the cytoplasm.