Post-transcriptional regulation of ribosome biogenesis in yeast

Most microorganisms are exposed to the constantly and often rapidly changing environment. As such they evolved mechanisms to balance their metabolism and energy expenditure with the resources available to them. When resources become scarce or conditions turn out to be unfavourable for growth, cells reduce their metabolism and energy usage to survive. One of the major energy consuming processes in the cell is ribosome biogenesis. Unsurprisingly, cells encountering adverse conditions immediately shut down production of new ribosomes. It is well established that nutrient depletion leads to a rapid repression of transcription of the genes encoding ribosomal proteins, ribosome biogenesis factors as well as ribosomal RNA (rRNA). However, if pre-rRNA processing and ribosome assembly are regulated post-transcriptionally remains largely unclear. We have recently uncovered that the yeast Saccharomyces cerevisiae rapidly switches between two alternative pre-rRNA processing pathways depending on the environmental conditions. Our findings reveal a new level of complexity in the regulation of ribosome biogenesis

FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins

Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae

Tor1 and CK2 kinases control a switch between alternative ribosome biogenesis pathways in a growth-dependent manner.

Ribosome biogenesis is a major energy-consuming process in the cell that has to be rapidly down-regulated in response to stress or nutrient depletion. The target of rapamycin 1 (Tor1) pathway regulates synthesis of ribosomal RNA (rRNA) at the level of transcription initiation. It remains unclear whether ribosome biogenesis is also controlled directly at the posttranscriptional level. We show that Tor1 and casein kinase 2 (CK2) kinases regulate a rapid switch between a productive and a non-productive pre-rRNA processing pathways in yeast. Under stress, the pre-rRNA continues to be synthesized; however, it is processed differently, and no new ribosomes are produced. Strikingly, the control of the switch does not require the Sch9 kinase, indicating that an unrecognized Tor Complex 1 (TORC1) signaling branch involving CK2 kinase directly regulates ribosome biogenesis at the posttranscriptional level

The switch from the A2 to the A3 pathway is not dependent on RNA pol I.

<p>(A) Northern blot analysis of the pre-rRNA processing in the strain NOY892, grown in YP-galactose. Hybridization probes as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.g001" target="_blank">Fig 1A and 1B</a>. (B) NOY892 was treated with 200 ng/ml rapamycin at OD₆₀₀ = 2 or exposed to heat shock by shifting from 25°C to 37°C. (C) Northern blot analysis of pre-rRNA processing in the anchor-away strain BEN135, expressing Rpa135–FRB fusion treated with rapamycin at OD₆₀₀ = 2. (D) Northern blot analysis of the pre-rRNA processing in the CARA strain treated with rapamycin OD₆₀₀ = 2.</p

Protein phosphorylation changes during diauxic shift.

<p>(A) Phosphorylation sites changed more than 2-fold during diauxic shift. The phosphorylation changes are represented by colored bars. The black arrows indicate sites with the CK2 kinase’s phosphorylation consensus sequence. (B) Phosphorylation sites changed >1.8-fold after TBB treatment. The black arrows indicate sites with the CK2 kinase’s phosphorylation consensus sequence. Underlying numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.s012" target="_blank">S1 Data</a>. (C) Updated model of pre-RNA processing in yeast.</p

TOR pathway controls the switch between the A2 and A3 pathways.

<p>(A) Pre-rRNA processing in YMK118 grown in YPD (OD₆₀₀ = 1.7) and treated with either DMSO or rapamycin, analyzed by northern blotting using probe A2–A3. (B) A rapamycin-insensitive <i>tor1-1</i> strain was treated the same as in (A). (C) Comparison of iBAQ values (normalized to bait) of 350 proteins detected in the affinity-purified preribosomes from cultures treated with DMSO or rapamycin. Proteins changed more than 2-fold are in black. (D) Comparison of SILAC H/L ratios (normalized to bait) of proteins from the affinity-purified preribosomes affected either by diauxic shift or by rapamycin treatment. (E) Histogram of SILAC H/L ratios (normalized to bait) for proteins with more than 2-fold change in the preribosomes purified from rapamycin versus DMSO-treated cultures. An experiment with a higher coverage is shown. Underlying numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.s012" target="_blank">S1 Data</a>.</p

TBB induces a switch between A2 and A3 pathways.

<p>(A) Strain YMK118 was treated with the CK2 kinase inhibitor TBB at OD₆₀₀ = 2, and samples were harvested at indicated times. Northern blotting using the probe A2–A3. (B) YMK118 was sequentially treated with inhibitors at OD₆₀₀ = 2 as indicated for 30 min. Then, a second inhibitor was added, and samples were harvested at indicated times. Total RNA was analyzed by northern blotting (probe A2–A3). (C) Histogram of SILAC H/L ratios (normalized to bait) of proteins with >2-fold change in preribosomes from TBB treated cells. The average of two experiments is shown. Underlying numerical data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.s012" target="_blank">S1 Data</a>.</p

Pre-rRNA processing switches between the A2 and A3 pathways.

<p>(A) A scheme of pre-rRNA processing in yeast, simplified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000245#pbio.2000245.ref022" target="_blank">22</a>]. Positions of oligonucleotides used as hybridization probes are indicated above. (B) Northern blot analysis of the pre-rRNA processing during different stages of growth. Wild-type yeast YMK118 was grown in YPD for up to 5 d. A growth curve with OD₆₀₀ values plotted against time is shown. An evaporation of culture media during extended cultivations likely affected the OD₆₀₀ reading. From our measurement, the error was <20% on day 5. The arrows indicate the time of the switch in pre-rRNA processing. Total RNA was isolated from the same number of cells and analyzed by northern blotting. The probes used for hybridization are indicated left of the gels. The mature 25S and 18S rRNAs were stained with Methylene blue after blotting to a nylon membrane. (C) YMK118 was grown in YPD (2% glucose). After yeast entered the postdiauxic phase, 1% glucose was added (arrow). Samples were harvested at indicated times. Total RNA was analyzed by northern blotting, using the A2–A3 probe. (D) Nitrogen limitation: YMK118 was grown in synthetic dextrose complete (SDC) media with limiting amounts of ammonium sulphate (625 mg/L, i.e., 8-fold less than in standard SDC media). Total RNA was analyzed as in (C). (E) Amino acid starvation; wild-type strain YMK118 was grown in SDC with limiting amounts of histidine (“low His”) (2 mg/L, i.e., 10-fold less than standard SD media). (F) Environmental stress: exponentially growing YMK118 was shifted from 25°C to 37°C or exposed to 0.2 mM Diamide. Total RNA was harvested at indicated times and analyzed as in (C).</p