Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae

Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

concentration of 6%, followed by incubation on ice for 1 h and centrifugation at 15,000 g for 15 min at 4 o C. The pellet obtained was suspended in Tris-buffered saline and counted in a liquid scintillation counter (Perkin Elmer Tri-carb 2900). The cpm (counts per min) values obtained were plotted using GraphPad Prism.
Ribosome profiles: Ribosome profiles were generated as described earlier [9] with some modifications. Yeast were grown in YPD till mid-log phase and were treated with cycloheximide (50 μg/mL), chilled on an ice-salt bath for 2-5 min and centrifuged immediately at 4,000 g. Cells were lysed in 1 mL lysis buffer (10 mM Tris pH 7.4, 100 mM NaCl, 30 mM MgCl 2 , 50 μg/mL cycloheximide, 200 μg/mL heparin, in 0.2% diethyl pyrocarbonate (DEPC)-treated water) and centrifuged at 10,000 g for 10 min at 4°C. Cell lysates equivalent to 10 A 254 units were loaded on top of a 10%-50% sucrose continuous gradient in buffer (50 mM Tris-HCl pH 7.4, 50 mM NH 4 Cl, 12 mM MgCl 2, 1 mM DTT, 0.1% DEPC) and centrifuged at 100,000 g at 4°C for 6 h in an SW41 rotor (Beckman). Ribosome levels were measured by gradient analysis on an ISCO UV-6 gradient collector by monitoring absorbance at 254 nm. To analyse individual ribosome subunits, lysates were resolved on a 10%-30% sucrose continuous gradient in buffer lacking MgCl 2 .

Doubling time and viability assessment:
Overnight grown yeast were sub-cultured in SC medium or in YPD at 0.1 OD 600 . Growth was monitored for 72 h by measuring OD 600 of the culture at regular intervals, and doubling time was calculated from the exponential phase of growth by linear regression analysis on a semi-logarithmic scale, using GraphPad Prism. To determine yeast cell mass, cells equivalent to 5 OD 600 were harvested from mid-log and stationary phase cultures, and washed with PBS. Cell pellets were dried at 50°C for 20 min and the dry weight of yeast was measured. To assess the cell number, cells in mid-log or stationary phase were counted using a Neubauer chamber and the number of cells present in 1 OD 600 was calculated. Cell death was monitored by incubating yeast cells in 0.2% trypan blue solution (Sigma-Aldrich) for 10 min, and scoring dead cells that take up the dye. To monitor cell viability, cells equivalent to 10 -5 OD 600 from mid-log and stationary phase cultures were plated on YPD-agar, incubated at 30°C for 48 h, and colonies were counted to extrapolate viable cell count per OD 600 .

RNA extraction and analysis:
Total RNA was isolated by hot phenol extraction as described earlier [10] with slight modifications. 1 OD 600 unit of cells from mid-log phase yeast cultures grown in YPD were lysed in AE solution (50 mM CH 3 COONa pH 5.3, 10 mM EDTA), containing 1% SDS and an equal volume of acid-buffered phenol, pH 4.3, followed by incubation at 65°C for 15 min with continuous shaking. Lysates were chilled on ice and centrifuged at 12,000 g for 10 min. The aqueous phase was transferred to a tube containing an equal volume of chloroform, mixed well and centrifuged at high speed. RNA was precipitated by the addition of 50 μL 3M sodium acetate followed by 100% ethanol, and dissolved in DEPC-treated water. RNA was estimated by measuring A 260 using a spectrophotometer (Thermo Scientific ND-1000). To monitor rRNA levels, 10µg of total RNA from each strain was resolved on a 1.2% formaldehyde-agarose gel.
RNA labelling experiments were performed by harvesting mid-log phase yeast cells grown in YPD. Cells equivalent to 1 OD 600 unit were incubated in SC-Ura medium containing 3 μCi/mL [ 14 C]uracil for different lengths of time, and RNA was extracted as described previously. Equal total RNA was resolved on a formaldehyde agarose gel, stained with ethidium bromide and transferred to an N + Hybond membrane (GE Life Sciences). Radiolabeled rRNA was detected using a phosphorimager scanner (Fujifilm FLA-9000). Pulse-chase analysis of rRNA was performed as described earlier [9], with slight changes. Yeast cells were harvested at an OD 600 of 0.5-0.7. The cells were washed and labeled in 1 mL SC-Ura medium containing 3 μCi/mL [ 14 C]uracil for 5 min at 30°C. A chase was performed with SC medium containing 240 mg/L unlabeled uracil. Samples were harvested 0, 1, 5, 15 and 20 min after the chase, and centrifuged at 12,000 g for 1 min at 4°C. RNA was extracted, and incorporation of radioactivity was detected as described earlier.

ChIP:
The assay was performed as described earlier [11] with slight modifications. 45 mL of mid-log phase yeast cultures grown in YPD were subjected to cross linking with 1% formaldehyde for 15 min at room temperature. Cross linking was quenched by adding glycine to a final concentration of 0.1 M. Cells were washed in ice cold Tris-buffered saline and were lysed in 500 μL of ice cold lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1 % sodium deoxycholate, 1 mM EDTA, protease inhibitor cocktail) by bead beating. Chromatin was fragmented using a bath sonicator (Diagenode). Cell lysates were centrifuged at high speed and the supernatant was pre-cleared with normal rabbit IgG followed by Protein A beads (GE Life Sciences). Supernatant was collected and 10 μL of this lysate was taken as input. Immunoprecipitation of chromatin was performed by incubating the lysate with anti-GST antibody overnight at 4°C followed by Protein A beads for 4 h. Beads were washed twice each in wash buffer I (50 mM HEPES pH 7.5, 500 mM NaCl, 1% Triton X-100, 0.1 % sodium deoxycholate, 1 mM EDTA, protease inhibitor cocktail), wash buffer II (10 mM Tris-HCI pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.75% NP-40, 0.75% sodium deoxycholate), and TE buffer. Chromatin was eluted in 100 μL of elution buffer (50 mM Tris-HCI pH 8.0, 10 mM EDTA, 1% SDS) and incubated at 65 o C overnight to reverse the cross linking. DNA was extracted using a PCR purification kit (Qiagen). PCR reactions were set up with primers 5'GCTAAGATTTTTGGAGAATAGC3' and 5'GCCTACTCGAATTCGTTTCC3' to amplify the rDNA promoter, and primers 5'TCAAACGGTGGAGAGAGTCG3' and 5'ACCAATGGAATCGCAAGATGC3' to amplify the 5'ETS. Real-time PCR was performed using Mesa Green 2X PCR MasterMix (Eurogentec) in a 20 μL reaction volume using 1 μL from the input sample and 3 μL from the immunoprecipitated sample (Applied Biosystems). Ct values of the immunoprecipitated samples were normalised to the adjusted Ct values of input, and data were plotted using GraphPad Prism.  Table S2. Primers used in this study to clone RNA Pol I subunits in pYesGex6p2 yeast expression vector [16]. *Inserts generated by overlap-extension PCR.

Plasmid Primers used to amplify insert pYesGex6p2
Yeast expression vector [16] Uaf30FL Uaf30  Table S3. Doubling time of yeast strains. Growth of the indicated yeast strains at 30°C in rich medium (YPD), synthetic complete (SC) medium or SC medium without uracil (SC-Ura) was monitored up to 48 h by measuring OD 600 of the culture at regular intervals. The doubling time was calculated from the logarithmic phase of growth. Data are mean ± SEM (n=4).