Ribosome profiling of synchronous, non-arrested yeast cells identifies translational control of lipid biosynthesis enzymes in the cell cycle

The translational efficiency of mRNAs in cells progressing synchronously through the mitotic cell cycle and having to meet their growth requirements for cell division has not been interrogated. Here, we used ribosome profiling of a cell-size series from the time of cell birth, to identify for the first time yeast mRNAs under periodic translational control. Late in the cell cycle, there was coordinate translational activation of mRNAs encoding lipid biosynthesis enzymes. An upstream open reading frame (uORF) mediates the translational control of ACC1, the mRNA encoding the rate-limiting enzyme acetyl-CoA carboxylase. The ACC1 uORF also adjusts Acc1p protein levels in different nutrients. Mutating the ACC1 uORF delayed cell cycle progression, reduced cell size and suppressed the replicative longevity of cells lacking the Sch9p protein kinase, the yeast S6 kinase ortholog. These findings reveal unexpected links between lipogenesis and protein synthesis in mitotic cell divisions We used centrifugal elutriation to isolate growing, highly synchronous, unbudded, early G1 cell populations and scored the elutriated cultures over time microscopically, recording the percentage of budded cells with a particle counter that directly measures their volume (channelyzer). Generation of both the Ribosome Profile (RP) and Transcriptional Profile (TP) libraries require at least ≥2E+09 yeast cells per sample. Such sample size could not be collected following a typical time-series experiment because only a small minority of the total cell population is in early G1 at any given moment. Instead, we allowed the entire cell population of each elutriated fraction to progress in the cell cycle until it reached a given cell size (e.g., 40 fl), at which point the sample was harvested. Elutriated cells of the same size were pooled together until enough cells per sample were obtained. This pool of same-size cells defined one experimental replicate (e.g., Sample01A). We harvested cells with 5fl cell size increments, from the time they are born (40 fl -- Sample01), until the end of the cell cycle (75 fl -- Sample08). In total we generated three independent Experimental Replicates for each cell size (A, B and C). We have: Sample01A, 01B and 01C (40 fl); Sample02A, 02B and 02C (45 fl), Sample03A, 03B and 03C (50 fl), Sample04A, 04B and 04C (55 fl), Sample05A, 05B and 05C (60 fl), Sample06A, 06B and 06C (65 fl), Sample07A, 07B and 07C (70 fl) and Sample08A, 08B and 08C (75 fl). Samples are divided into two series: Ribosome Profile (RP) and Transcriptional Profile (TP).