An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Abstract

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s(2)U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s(2)U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s(2)U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.

Keywords: URM1; evolution; tRNA modification; temperature stress; yeast.

FIGURE 1.

Saccharomyces cerevisiae tolerates elevated temperatures. Serial dilutions (1:5) of S. cerevisiae S288C and W303 were spotted onto rich growth medium (YPD) plates and incubated for 3 d at the indicated temperatures. Note the absence of growth for both strains at 41°C and 43°C.

FIGURE 2.

Quantitative liquid chromatography mass spectrometry analysis reveals differences in tRNA modification levels following temperature stress. (A) Superimposed extracted-ion chromatograms (XICs) of the 18 most prominent nucleoside modifications found in yeast. The average retention time (n = 3) for each modification in this chromatographic setup is indicated in minutes: 1, D (13.8); 2, Ψ (17.37); 3, ncm⁵U (29.03); 4, m³C (30.05); 5, m¹A (31.78); 6, m⁵C (34.57); 7, m⁷G (36.24); 8, Cm (36.75); 9, I (38.28); 10, m⁵U (39.32); 11, t⁶A (43.44); 12, m¹I (44.82); 13, mcm⁵U (45.37); 14, m¹G (45.75); 15, ac⁴C (46.53); 16, m²G (46.63); 17, m^2,2G (48.83); 18, Am (51.65). In addition, we detect yW (26.68), mcm⁵s²U (39.43), Um (42.06), ct⁶A (44.44), Gm (46.93), ncm⁵Um (47.92), and i⁶A (55.50). The canonical bases are omitted for clarity. (B) Full mass spectrum depicting the neutral loss occurring upon ionization-induced fragmentation of a nucleoside with a native (m¹A) or methylated (Cm) ribosyl group, respectively. (C) Heat map depicting the relative comparison of normalized modification levels at various temperatures for 21 nucleosides detected by pyQms in samples from S. cerevisiae S288C and W303. The scale bar indicates the fold change in modification levels compared with 30°C (10-fold increase shown as white, no change as pale orange, and 100-fold decrease as black). Modifications that yield a weak MS signal are labeled in gray.

FIGURE 3.

2-Thiolation levels in S. cerevisiae respond to elevated temperatures. (A,B) Representative autoradiogram of tRNA extracted at different growth temperatures from (A) S288C and (B) W303 following ([N-acryloylamino]phenyl)mercuric chloride (APM)-affinity gel electrophoresis and Northern blot with the tK^UUU probe. The horizontal arrows in this and subsequent Northern blot images mark thiolated (black) and non-thiolated (gray) tRNA species. 2-Thiolation levels were quantified for all three tRNA isoacceptors (n = 3, apart from W303 samples grown at 25°C where n = 2). (C) Superimposed UV-chromatograms from UPLC analysis of enzymatically digested tRNA of S288C. The position of the canonical bases is indicated, and synthetic standards of mcm⁵U (20.3 min) and mcm⁵s²U (31.5 min; boxed) were used to determine the average retention times (n = 3). Note the decreasing height and area of the mcm⁵s²U peak at elevated temperatures. The relative change in peak area for mcm⁵s²U is depicted (normalized to 30°C).

FIGURE 4.

Temperature-induced reduction of tRNA 2-thiolation is reversible. (A) Scheme of the time course assay. The 37°C culture is shown as a light gray line; the culture at 30°C is depicted by a dark gray line (for this and all subsequent figures). Arrows indicate time points for sample collection. (B) Representative autoradiogram following APM-affinity gel electrophoresis and Northern blot with tK^UUU probe of tRNA collected at the indicated time points. 2-Thiolation levels were quantified by image densitometry (n = 3).

FIGURE 5.

Inhibition of tRNA synthesis stalls 2-thiolation. (A) Growth curve measurements of S288C cells in logarithmic growth at 37°C supplemented with RNA polymerase inhibitor ML-60218 (top panel) or thiolutin (middle panel) at the 0 h time point. rpo31-698 was grown at 30°C and shifted to 37°C at the 0 h time point (bottom panel). (B) Representative autoradiogram of tRNA extracted at indicated time points from ML-60218 treated cells following APM-affinity gel electrophoresis and Northern blot with tE^UUC probe. Arrowheads highlight the 0 h time point at which the inhibitor was added. Thiolation levels were quantified by image densitometry (n = 3). (C) As in B for thiolutin. (D) As in B for rpo31-698, where inhibition of tRNA synthesis is induced by the temperature shift to 37°C.

FIGURE 6.

Evolutionary divergent yeasts have different temperature preferences. (A) Phylogenetic relationship and evolutionary distance of all yeast species included in this study. (Data adapted from Rhind et al. 2011.) (B) Serial dilutions (1:5) of five yeast species were spotted onto rich growth medium (YPD or YES) plates and incubated for 3 d at the indicated temperatures.

FIGURE 7.

2-Thiolation levels are not affected by temperature in most other yeast species. Representative autoradiogram images of tRNA extracted at different growth temperatures from (A) S. bayanus, (B) S. mikatae, (C) S. paradoxus, (D) C. glabrata, and (E) S. pombe following APM-affinity gel electrophoresis and Northern blot with the tK^UUU probe. 2-Thiolation levels were quantified by image densitometry (n = 3, apart from samples grown at 25°C where n = 2). Asterisks (*) show unspecific binding of the probe.

FIGURE 8.

tRNA modification landscapes for divergent yeast species show surprising similarity. Heat map of the relative change in nucleoside modification levels compared with 30°C in S. bayanus, S. mikatae, S. paradoxus, C. glabrata, and S. pombe using pyQms. The scale bar shows a 10-fold increase in modification levels as white, no change as pale orange, and a 31.6-fold decrease as black. Modifications that yield a weak MS signal are labeled in gray.