A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis
- Institute for Stem Cell Science and Regenerative Medicine (inStem), India
- Rutgers University, United States
- Manipal Academy of Higher Education, India
Figures
Figure 1 with 2 supplements
Amino acid and nucleotide metabolism are decoupled in tRNA thiolation deficient cells.
(A) Intracellular pools of amino acids are increased in tRNA thiolation mutants. Steady-state amino acid amounts were measured in wild-type (WT) and tRNA thiolation mutant cells (uba4Δ and ncs2Δ) …
Figure 1—figure supplement 1
Gcn2-dependent translational induction of Gcn4 in tRNA thiolation deficient cells.
(A) GCN4 translation is increased in tRNA thiolation mutants. A schematic representation of different Gcn4-luciferase (Gcn4-luc) translational reporter constructs. Two upstream ORFs in the 5’ UTR of …
Figure 1—figure supplement 2
Amino acid and nucleotide measurements in tRNA thiolation deficient cells.
(A) Intracellular levels of nucleotides are decreased in tRNA thiolation mutants. Steady-state nucleotide (AMP, GMP, CMP and UMP) amounts were measured in wild-type (WT) and tRNA thiolation mutant (u…
Figure 2 with 2 supplements
Carbon flux is routed towards storage carbohydrates in thiolation mutants.
(A) Schematic representation depicting nucleotide and storage carbohydrates (trehalose and glycogen) biosynthesis, starting from a common precursor, glucose-6-phosphate. In normal conditions where …
Figure 2—figure supplement 1
Carbon flux is routed away from nucleotides and towards storage carbohydrates in tRNA thiolation deficient cells.
(A) Nucleotide synthesis is decreased in tRNA thiolation mutants. Wild-type (WT) and tRNA thiolation mutant cells (ncs2Δ) grown in minimal media were pulse-labelled with [U-13C6]-labelled glucose …
Figure 2—figure supplement 2
Sulfur starvation phenocopies the metabolic state of thiolation mutants.
(A) A schematic representation illustrating the experimental design for sulfur starvation. Wild type cells growing in standard glucose medium were shifted to sulfur-rich and sulfur-starved/sulfur …
Figure 3 with 1 supplement
tRNA thiolation couples cellular metabolic state with normal cell cycle progression.
(A) tRNA thiolation mutants exhibit asynchronous cell division and disrupted yeast metabolic cycles. WT and tRNA thiolation mutant cells (uba4Δ) growing in chemostat cultures under conditions of …
Figure 3—figure supplement 1
HU sensitivity in tRNA thiolation deficient cells is independent of the Rad53 checkpoint pathway.
(A) Catalytically dead uba4 mutants (C225A and C397A) exhibit increased HU sensitivity. Wild-type (WT), tRNA thiolation mutant cells (uba4Δ and ncs2Δ) and catalytically dead uba4 mutant (C225A and …
Figure 4 with 3 supplements
Loss of tRNA thiolation results in reduced phosphate homeostasis (PHO)-related transcripts and ribosome-footprints.
(A) Correlation plots are shown, for WT and tRNA thiolation mutant cells (ncs2Δ and uba4Δ), for both gene expression (transcript) and ribosome footprint (translation) changes. The coefficients of …
Figure 4—figure supplement 1
Transcript and ribosome footprint profiles in wild-type and tRNA thiolation mutant cells.
(A) Correlation plots, for RNA (transcript) or ribosome footprint (RPF), between biological replicates, of WT, or thiolation mutant cells. Note, correlation coefficients (R) for any replicate of the …
Figure 4—figure supplement 2
Gene expression (transcript) and ribosome footprint (translation) changes for wild-type (WT) and tRNA thiolation mutant cells.
(A) Changes in the ribosome density based on codon occupancy are shown at A, P, and E sites. Different codons are highlighted in both plots. n = 3 biological replicates for each strain are plotted. …
Figure 4—figure supplement 3
Gene expression and ribosome footprint changes for wild-type (WT) and tRNA thiolation mutant cells in PHO regulon, ribosomal subunits, PPP, trehalose/glycogen, nucleotide and amino acid synthesis genes.
(A) Heat maps representing changes in expression (transcript and ribosome footprints) of genes involved in nucleotide biosynthesis, for wild-type (WT) and tRNA thiolation mutant cells (uba4Δ and ncs2…
Figure 5 with 1 supplement
tRNA thiolation mutants exhibit a dampened PHO response.
(A) A schematic representation of PHO regulon-related genes, and their roles. Pho84 and Pho89 are high affinity phosphate transporters, Pho87 and Pho90 are low affinity phosphate transporters, Spl2 …
Figure 5—figure supplement 1
tRNA thiolation mutants exhibit a dampened PHO response.
(A) Pho12 protein is decreased in tRNA thiolation mutants in high and low Pi conditions. Using cells expressing endogenous Pho12 with a FLAG epitope at its carboxy-terminus, Pho12 protein levels in …
Figure 6 with 1 supplement
Phosphate depletion in wild-type cells phenocopies tRNA thiolation mutants.
(A) Trehalose amounts are increased upon phosphate starvation. Trehalose content of WT cells grown in high and no Pi media was plotted. Data are displayed as means ± SD, n = 3. ****p<0.0001. (B) …
Figure 6—figure supplement 1
Gcn2-dependent translational induction of Gcn4 during phosphate starvation.
(A) GCN4 translation is Gcn2-dependent in phosphate-starved wild-type cells. Wild-type (WT) and gcn2Δ cells transformed with the WT Gcn4-luc construct were grown in high and no phosphate medium …
Figure 7 with 1 supplement
Trehalose synthesis associated phosphate release enables cells to maintain phosphate balance.
(A) A schematic representation of the trehalose cycle, showing the routing of glucose-6-phospate towards trehalose and glycogen biosynthesis. Trehalose synthesis requires two glucose molecules, …
Figure 7—figure supplement 1
Tps2 maintains phosphate balance in tRNA thiolation deficient cells.
(A) Intracellular Pi levels are maintained in tRNA thiolation mutant cells (uba4Δ and ncs2Δ) by Tps2 activity. Free intracellular Pi levels were determined in wild-type (WT), tRNA thiolation mutants …
Figure 8
A model illustrating how tRNA thiolation regulates the metabolic state of the cell.
WT cells (which have a functional tRNA thiolation machinery) in amino acid (methionine and cysteine) replete conditions have high amounts of thiolated tRNAs. In these cells, carbon flux coupled with …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-HA | Roche | Cat# 12CA5 | WB (1:2000) |
| Antibody | anti-phospho eIF2a (Ser51) | Cell Signalling Technology | Cat# 9721S | WB (1:1000) |
| Antibody | anti-FLAG | Sigma-Aldrich | Cat # F1804-5MG | WB (1:2000) |
| Antibody | anti-Rad53 yC-19 | Santa Cruz Biotechnology | Cat # sc-6749 | WB (>1:1000) |
| Antibody | HRP-conjugated secondary antibodies (anti-mouse, anti-rabbit) | Sigma-Aldrich | Cat # 7076S Cat # 7074S | WB (1:5000) |
| HPLC column | Synergi 4µ Fusion-RP 80A column (100 × 4.6 mm) | Phenomenex | Cat # 00D-4424-E0 | |
| Sequence-based reagent | Gcn4-luciferase translation reporters | this study | ||
| Peptide, recombinant protein | trehalase | Sigma-Aldrich | Cat # T8778 | |
| Peptide, recombinant protein | amyloglucosidase | Sigma-Aldrich | Cat # 10115 | |
| Peptide, recombinant protein | RNAse A | Sigma-Aldrich | Cat # R4875 | |
| Peptide, recombinant protein | protease solution | Sigma-Aldrich | Cat # P6887 | |
| Commercial assay or kit | Glucose estimation kit | Sigma-Aldrich | Cat # GAGO20 | |
| Commercial assay or kit | RiboZero | Epicenter | Cat # MRZH116 | |
| Commercial assay or kit | luciferase assay kit | Promega | Cat # E1500 | |
| Commercial assay or kit | Maxima SYBRGreen/ROX qPCR Master Mix | Thermo Scientific | Cat # K0222 | |
| Commercial assay or kit | ATP estimation kit | Thermo Scientific | Cat # A22066 | |
| Chemical compound, drug | SYTOX green | Thermo Scientific (Invitrogen) | Cat # S7020 | |
| Chemical compound, drug | p-nitrophenyl phosphate | Sigma-Aldrich | Cat # N4645 | |
| Chemical compound, drug | bicinchoninic acid assay | Thermo Scientific | Cat # 23225 | |
| Software, algorithm | Prism 7 | Graphpad |
Additional files
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Supplementary file 1
Strains used in this study.
- https://doi.org/10.7554/eLife.44795.022
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.44795.023
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Supplementary file 3
Mass transitions for detection of metabolites.
- https://doi.org/10.7554/eLife.44795.024
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Supplementary file 4
RNA-RFP changes (mutant-WT).
- https://doi.org/10.7554/eLife.44795.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.44795.026
