Figure 1. tRNA uridine thiolation levels reflect sulfur amino acid availability.
(A) tRNA structure, wobble uridine modifications, and structures of methoxycarbonylmethyl (mcm5) and methoxycarbonylmethyl + thiolation (mcm5s2) uridine modifications.
(B) Experimental approach for measuring tRNA uridine modifications by LC-MS/MS.
(C) tRNA uridine thiolation amounts decrease in minimal media. Relative abundance of thiolated (mcm5s2) or mcm5-modified tRNA uridines in cells grown in rich (YPD, YPL) and minimal media (SD, SL) were measured. (Mean±SD, n=4, with experimental duplicates. *** indicates p< 0.001, ** indicates p<0.01, * indicates p< 0.05). See also Figure S1.
(D) tRNA uridine thiolation amounts are rescued by sulfur-containing amino acids. Cells were grown as described in (B), with or without supplementation of the indicated amino acids, ammonium sulfate (NH4)2SO4, or a non-sulfur amino acid mixture (non-S). Tyrosine was excluded due to poor solubility. (Mean±SD, n=3, with experimental duplicates). See also Figure S1.
(E) Simplified schematic of sulfur amino acid metabolism in budding yeast. Cysteine and methionine are derived from homocysteine, and can be interconverted.
(F) Intracellular sulfur amino acid levels decrease in SD minimal medium. Cells were grown in YPD, SD, or SD supplemented with methionine (150 μM) (see panel B), and sulfur metabolites were measured by LC-MS/MS. Note: relative decreases of both cysteine and methionine in SD mirror the decrease in uridine thiolation abundance. (Mean±SD, n=4, with experimental duplicates). See also Table S1 and Figure S1.