Fig 1.
Pre-rRNA processing switches between the A2 and A3 pathways.
(A) A scheme of pre-rRNA processing in yeast, simplified from [22]. 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).
Fig 2.
In vivo pulse-chase and proteomic analyses of the pre-rRNA processing in exponential and postdiauxic shift cultures.
(A) Low density (OD600 = 2) YMK118 was pulse labeled in vivo with 3H-uracil for 6 min and then chased with nonradioactive uracil for 10 min. (B) Yeast culture in early postdiauxic phase (OD600 = 10) was pulse labeled for 6 min. The culture was split, and one-half was chased for 10 min, as in (A) (on the left). To the other half of the culture, fresh media and no-radioactive uracil was added to the same final concentration and chased for 10 min (right side, “exponential chase”). (C) Comparison of intensity based absolute quantification (iBAQ) values normalized to bait of 324 proteins detected in the affinity purified preribosomes from pre- and postdiauxic cultures. Proteins changed more than 2-fold are in black. (D) Histogram of SILAC Heavy/Light (H/L) ratios (normalized to bait) for proteins with more than 2-fold change in the preribosomes purified from postdiauxic shift versus prediauxic shift cultures (for high resolution see S7 Fig). An experiment with a higher coverage is shown. Underlying numerical data are in S1 Data. (E) Northern blot analysis of RNA isolated form wild-type yeast or strains deleted for different exosome/TRAMP complex factors or Xrn1. All strains were grown until their growth stopped after the diauxic shift. The xrn1Δ strain stopped growing at the indicated lower OD600 than the other strains.
Fig 3.
TOR pathway controls the switch between the A2 and A3 pathways.
(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 tor1-1 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 S1 Data.
Fig 4.
The switch from the A2 to the A3 pathway is not dependent on RNA pol I.
(A) Northern blot analysis of the pre-rRNA processing in the strain NOY892, grown in YP-galactose. Hybridization probes as described in Fig 1A and 1B. (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.
Fig 5.
Role of Sch9, tap42, or tip41 in the A2 to A3 switch in pre-rRNA processing.
(A) Pre-rRNA processing before and after diauxic shift in the yeast strain lacking Sch9 kinase (sch9Δ) or in the tap42-11 mutant. The OD600 values of harvested samples are shown above the lanes. Northern blot using the A2–A3 probe. (B) Pre-rRNA processing before and after diauxic shift in the strain expressing the hyperactive mutant Sch9-2D3E grown in SDC media. Samples were harvested at indicated OD600, and total RNA was analyzed by northern blotting using probe A2–A3. (C) The sch9Δ or wild-type strains were treated by rapamycin at OD600 = 2, and total RNA was isolated at indicated time points and analyzed by northern blotting (probe A2–A3). (D) The tap42-11 mutant and tip41Δ strain were treated with rapamycin as in (C).
Fig 6.
TBB induces a switch between A2 and A3 pathways.
(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 S1 Data.
Fig 7.
Protein phosphorylation changes during diauxic shift.
(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 S1 Data. (C) Updated model of pre-RNA processing in yeast.