Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul 24;290(30):18699-707.
doi: 10.1074/jbc.M115.665406. Epub 2015 Jun 10.

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

Diego Rojas-Benitez  1 , Patrick C Thiaville  2 , Valérie de Crécy-Lagard  2 , Alvaro Glavic  3
Affiliations

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

Diego Rojas-Benitez et al. J Biol Chem. .

Abstract

N(6)-Threonylcarbamoyl-adenosine (t(6)A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNAi (Met)). Yeast lacking central components of the t(6)A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t(6)A synthesis. In Drosophila and in mammals, tRNAi (Met) is a limiting factor for cell and animal growth. We report that the t(6)A-modified form of tRNAi (Met) is the actual limiting factor. We show that changing the proportion of t(6)A-modified tRNAi (Met), by expression of an un-modifiable tRNAi (Met) or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t(6)A-modified tRNA, determines growth potential in eukaryotic cells.

Keywords: Drosophila; cell growth; transfer RNA (tRNA); translation; yeast.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
tcs3 mutants exhibit severe growth phenotypes. A, schematic representation of anti-codon stem loop of tRNAs that pair A-starting codons, indicating position 37 where t6A is present. Anti-codon (red) and codon (black) are also depicted. B, comparison between larvae from wild-type (wt), homozygous mutants for tcs3 (tcs3f and tcs3e) and a trans heterozygous genetic background (tcs3f/tcs3e). Ubiquitous Tcs3 expression using the armadillo-Gal4 driver (tcs3e+Tcs3) rescued the growth phenotype. C, Tcs3 mRNA was not detected by RT-PCR in tcs3f. Actin was amplified as loading control. D, mosaic fat body generated by FRT/FLP-mediated recombination shows that tcs3 is required for growth cell autonomously. Cell area quantification shows that mutant cells (GFP−, white arrowhead) are significantly smaller than wild-type cells (GFP+) (n = 50 cells).
FIGURE 2.
FIGURE 2.
Tcs3 is required for t6A synthesis in Drosophila. A, yeasts were plated in solid media in serial dilutions of 1:10 factor from left to right. Growth differences of wild-type, TCS3 mutant cells (Δtcs3), and mutant yeasts expressing the Drosophila Tcs3 homolog (Δtcs3+Tcs3). Growth parameter of each strain was also analyzed in liquid media. Each strain has a color code (n = 10, p < 0.005). B, in Drosophila, Tcs3 was knocked-down using a specific inverted repeat construct expressed in the posterior wing compartment with the Gal4/UAS system (Tcs3-IR) and the growth phenotype evoked was rescued by yeast Tcs3p co-expression (en> Tcs3-IR+Tcs3). The area of sector D (colored in green) was measured and plotted following the same color code used in wing image labels (n = 50, mean ± S.D., t test p < 0.005). C, PHAt6A. In brief, probes designed against different regions of the initiator tRNA evidenced the presence of t6A modification. The strength of ASL probe (anti-codon stem loop) hybridization depends on the presence of t6A, whereas the TΨL probe hybridizes an unmodified base stretch and serves as an internal loading control. When t6A is absent, the tRNA-probe interaction is maximal, whereas its existence weakens the interaction. D, tRNAs from wild-type (wt) and mutant tcs3 animals (tcs3e and tcs3f) were probed with ASL and TΨL probes. Dot blots were merged to make a better composition. Also a plot depicting the change in the ASL/TΨL signal ratio is shown. ASL signals obtained from tcs3 mutants are significantly stronger than control counterparts (n = 6 samples).
FIGURE 3.
FIGURE 3.
Expression of an Ala-37 mutant initiator tRNA reduces growth. A, comparison between wings from wild-type and from tRNAiMet(A37G) homozygous transgenic animals. A significant reduction in wing area, together with a higher cell density (B) is detected in transgenic animals compared with control wings (n = 50 wings).
FIGURE 4.
FIGURE 4.
An increment of t6A-modified tRNAs promotes growth cell-autonomously. A, representative image of PHAt6A assay in samples from control and Tcs3-overexpressing animals. Signal intensity was quantified and plotted as ASL/TΨL ratio (n = 4 samples). B, a comparison between control (black line) and Tcs3-overexpressing wings (red line). Wing area was plotted showing that Tcs3 significantly promotes growth (n = 50 wings). As controls we used two other UAS insertions located in the second and third chromosome (Tcs3(B) and Tcs3(C)) were able to promote animal growth. Likewise other interventions with known effects on animal growth are: Maf1 knockdown and the addition of an extra locus of tRNAiMet. Measurements of wing area from control (da>+) and experimental animals show that these manipulations favor animal growth in a comparable range (n = 50, p < 0.005 to control). C, flow cytometry was used to compare cell size in Tcs3-overexpressing and control cells. hedgehog-Gal4 driver (hh>Gal4) was used to express Tcs3 and GFP in the wing posterior compartment, whereas control cells were obtained from the anterior compartment of the same imaginal discs (no GFP). Histogram are representative of 3 independent experiments. D, FLP-out Tcs3-overexpressing mosaic analysis (bar = 100 μm). Control clones express GFP only, whereas Tcs3-overexpressing clones express Tcs3 and GFP. Clone area was measured and normalized by the area of its neighbor cells; these ratios are presented in the chart (n = 50 cells). Wild-type yeast strain (BY4741) was transformed with empty pDEST52 vector (control) or with a pDEST52 construct to overexpress the D. melanogaster Tcs3p coding sequence (+Tcs3p). E, plot representing an archetypal HPLC elution profile of nucleosides from control (black line) or Tsc3p-overexpressing yeast (red line). AU, arbitrary units. F, serial dilution assays (1:10) were made from a suspension of cells with A600 = 0.6. Growth assay in liquid media were made measuring simultaneously the A600 of 10 independent wells and the plot is representative of 3 independent experiments.
FIGURE 5.
FIGURE 5.
Variations of TOR activity in animals with different levels of t6A-modified tRNAs. A, Western blot of S6K and Akt phosphorylation performed by TORC1 and TORC2, respectively, which in tcs3 mutants (tcs3e and tcsf) was strongly reduced in comparison to wild-type animals. Total S6K, Akt, and actin were used as loading controls. Also we detected eIF2α phosphorylation at Ser-51 in these genetic backgrounds; actin was used as loading control. B, polysome profiles were constructed measuring 260 nm absorbance in samples obtained after ultracentrifugation in sucrose gradient. The polysome fraction (indicated with a black line) was identified in divalent cation-free extraction buffer and in the presence of chelating agents, wild-type (green) and tcs3e mutant (red) larvae. C, comparison between wild-type and tcs3e mutants expressing Rheb (tcs3e+Rheb) using the armadillo>Gal4 driver. D, Western blot detection of S6K phosphorylation in mutant (tcs3e) and mutant overexpressing Rheb (tcs3e+Rheb). E, Western blot detecting S6K phosphorylation in control (da>+) and Tcs3-overexpressing animals (da>Tcs3). Western blot images are representative of 3 independent experiments in each case.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Crick F. H. (1968) The origin of the genetic code. J. Mol. Biol. 38, 367–379 - PubMed
    1. Raina M., Ibba M. (2014) tRNAs as regulators of biological processes. Front. Genet. 5, 171. - PMC - PubMed
    1. Wek S. A., Zhu S., Wek R. C. (1995) The histidyl-tRNA synthetase-related sequence in the eIF-2α protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol. Cell. Biol. 15, 4497–4506 - PMC - PubMed
    1. Mei Y., Yong J., Liu H., Shi Y., Meinkoth J., Dreyfuss G., Yang X. (2010) tRNA binds to cytochrome c and inhibits caspase activation. Mol. Cell 37, 668–678 - PMC - PubMed
    1. Unger M. W., Hartwell L. H. (1976) Control of cell division in Saccharomyces cerevisiae by methionyl-tRNA. Proc. Natl. Acad. Sci. U.S.A. 73, 1664–1668 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources