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. 2015 Feb;21(2):188–201. doi: 10.1261/rna.048173.114

Functional importance of Ψ38 and Ψ39 in distinct tRNAs, amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

Lu Han 1, Yoshiko Kon 1, Eric M Phizicky 1,✉
PMCID: PMC4338347  PMID: 25505024

Abstract

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U38 and U39 in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ38 or Ψ39 is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNAGln(UUG), which normally has Ψ38. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s2U moiety of mcm5s2U34 of tRNAGln(UUG) and the other two cytoplasmic species with mcm5s2U34, that the reduced s2U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s2U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ38,39 provides evidence that Ψ38 is important for function of tRNAGln(UUG) at permissive temperature, and indicates that Ψ39 is important for the function of tRNATrp(CCA) in trm10Δ pus3Δ mutants and of tRNALeu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ38 and Ψ39 in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.

Keywords: PUS3, UBA4, KTI12, Saccharomyces cerevisiae, pseudouridine

INTRODUCTION

Modifications are universally found in tRNA molecules from all environmental niches examined, including the simplest organisms with the most streamlined genomes. These modifications are known to play important roles in ensuring the folding and stability of the tRNA (Helm et al. 1999; Kadaba et al. 2004; Alexandrov et al. 2006), high accuracy of charging (Muramatsu et al. 1988), maintenance of the correct reading frame (Urbonavicius et al. 2001), and accurate decoding of mRNAs (Murphy et al. 2004; Weixlbaumer et al. 2007). Modifications in and around the anticodon loop have profound roles in the cell. Several modifications found at the wobble residue N34 in the yeast Saccharomyces cerevisiae likely exert their biological effects through decoding. Mutants lacking I34 (inosine) are lethal (Gerber and Keller 1999), and trm4Δ mutants, which lack m5C (5-methylcytidine), are sensitive to oxidative stress (Chan et al. 2010). Similarly, mutations in the complex required for formation of the cm5U34 moiety of the xcm5U modifications (mcm5U, 5-methoxycarbonylmethyluridine; mcm5s2U, 5-methoxycarbonylmethyl-2-thiouridine; ncm5U, 5-carbamoylmethyluridine and ncm5Um, 5-carbamoylmethyl-2′-O-methyluridine) are temperature-sensitive (Jablonowski et al. 2001; Krogan and Greenblatt 2001) and have defects in transcription, exocytosis, silencing, and DNA damage response (Otero et al. 1999; Rahl et al. 2005; Li et al. 2009), all owing to two of the 11 tRNAs with these modifications (Esberg et al. 2006; Chen et al. 2011).

Modifications at other positions of the anticodon stem–loop also affect function in S. cerevisiae. At residue 37, mutants lacking t6A37 (N6-threonlycarbamoyladenosine) (El Yacoubi et al. 2011; Srinivasan et al. 2011) or m1G37 grow very poorly (Björk et al. 2001), and lack of i6A37 (N6-isopentenyladenosine) results in reduced nonsense suppression (Dihanich et al. 1987) and tRNA gene-mediated silencing (Pratt-Hyatt et al. 2013), as well as increased resistance to certain antifungal drugs (Suzuki et al. 2012). Similarly, lack of Cm32 and Nm34 due to lack of TRM7 leads to poor growth because of reduced function of tRNAPhe (Pintard et al. 2002; Guy et al. 2012).

Pseudouridine is the most common modification found in tRNA from all domains of life, and Ψ38 and Ψ39 are two of the four most-conserved pseudouridine modifications (Charette and Gray 2000). Among characterized tRNA species in archaea, eukaryota, eubacteria, and viruses, Ψ is found in 37 of 56 tRNAs with U38, and in 163 of 178 tRNAs with U39 (Table 1; Juhling et al. 2009). Furthermore, Ψ39 is found in several tRNA species of the molliculite Mycoplasma capricolum, a bacterial species in the phlyum firmicutes with a streamlined genome and only 13 different tRNA modifications (Andachi et al. 1989). The family of genes encoding the pseudouridine synthase responsible for Ψ38 and Ψ39 modifications is also highly conserved (Koonin 1996; Mueller and Ferre-D'Amare 2009). In Escherichia coli and Salmonella typhimurium, TruA catalyzes formation of Ψ38, Ψ39, and Ψ40 (Singer et al. 1972; Hur and Stroud 2007), whereas in S. cerevisiae, the related Pus3 (Deg1) catalyzes Ψ38 and Ψ39 modification (Lecointe et al. 1998) and in Haloferx volcanii, HVO_1852 catalyzes Ψ39 and likely Ψ38 modification (Blaby et al. 2011). Furthermore, the TruA/Pus3 family has a homolog in the streamlined bacterial genomes of endosymbionts, derived from α and γ proteobacter, and in molliculites (de Crécy-Lagard et al. 2012).

TABLE 1.

The occurrence of Ψ38 and Ψ39 is conserved in all domains of life

graphic file with name 188TB1.jpg

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The TruA/Pus3 pseudouridylases are important, but not essential. Mutation of hisT (TruA), the pseudouridylase catalyzing formation of Ψ38–40 in E. coli and Salmonella typhimurium results in derepression of the histidine operon, and a modest-to-severe reduction in growth rate (Chang et al. 1971; Tsui et al. 1991), while S. cerevisiae pus3Δ mutants are distinctly slow growing (Carbone et al. 1991) and temperature-sensitive (Lecointe et al. 2002), and have reduced −1 frameshifting owing to lack of Ψ39 (Bekaert and Rousset 2005). Biochemical and structural analysis shows that Ψ stabilizes both duplex and single-stranded RNA in part owing to coordination of a water molecule through the N1H group of Ψ and the adjacent 5′ phosphates, and from enhanced stacking in both single-stranded and duplex helices owing to its favoring of 3′ endo conformation (Arnez and Steitz 1994; Davis 1995; Durant and Davis 1999; Charette and Gray 2000).

We investigated here the cause of the temperature sensitivity of pus3Δ mutants of S. cerevisiae. Our results demonstrate that although Ψ38 or Ψ39 are found on at least 19 tRNA species in yeast (all of the characterized species with U38 or U39), the primary defect at high temperature is due to poor function of tRNAGln(UUG). Surprisingly, we find that the defect in tRNAGln(UUG) is due to loss of both Ψ38 and s2U at high temperature, and that the loss of s2U occurs at high temperature in commonly used wild-type S. cerevisiae strains. Moreover, we provide evidence that Ψ38 has a role in tRNAGln(UUG) at low temperature, and that Ψ39 has a role in the function of tRNATrp(CCA) and in suppression by tRNALeu(CAA)am. Our results emphasize that Pus3 has distinct effects on specific tRNA species and demonstrate that relatively mild temperature changes can alter the modification spectrum of cellular tRNAs.

RESULTS

The temperature sensitivity of pus3Δ mutants is primarily due to the defect of tRNAGln(UUG)

To begin elucidating the important role of PUS3, we performed a screen for high copy suppressors of the temperature sensitivity of pus3Δ mutants. A reconstructed pus3Δ mutant strain was temperature-sensitive on rich (YPD) plates at 38°C and grew more poorly than wild type in liquid YPD media at 30°C and 37°C (Fig. 1A,B), essentially as previously reported (Carbone et al. 1991; Lecointe et al. 1998). Based on the temperature sensitivity on plates, we transformed 17 pools of plasmids from an arrayed library of ∼1700 high copy (2μ) plasmids with inserts spanning nearly the entire yeast genome (Jones et al. 2008), and plated pools of transformants at 38°C and 39°C in YPD. We identified two pools containing potential suppressors that did not contain PUS3, each of which after deconvolution had in common a tQ(UUG) gene (encoding tRNAGln(UUG)), which proved to be responsible for suppression of the temperature sensitivity when expressed on a 2μ plasmid containing no other genes (Fig. 1A). As the modifications of tRNAGln(UUG) are uncharacterized and there is an encoded uridine at residue 38, this uridine was a potential Pus3 substrate. Indeed, we found that after growth at 30°C, tRNAGln(UUG) purified from pus3Δ mutants had one less mol of Ψ than in wild-type cells (2.88 mol/mol compared with 3.73 mol/mol), whereas the levels of m1A and mcm5s2U were virtually unchanged in both strains (Fig. 1C).

FIGURE 1.

FIGURE 1.

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tRNAGln(UUG) is the major biologically important target of Pus3 at high temperature. (A) The pus3Δ mutant is temperature sensitive and is partially suppressed by overproduction of tRNAGln(UUG). Wild-type and pus3Δ strains containing [2μ LEU2] plasmids as indicated were grown overnight in SD-Leu medium at 30°C, adjusted to OD600 of ∼0.8, and serially diluted, and then 2 μL was spotted onto YPD plates and incubated at the indicated temperature for 2 d. (B) The pus3Δ mutant grows more poorly than wild type in liquid medium at different temperatures. pus3Δ and wild-type strains were grown overnight in YPD medium at 30°C, diluted in prewarmed YPD medium at the indicated temperature, and growth was monitored. (C) tRNAGln(UUG) is a substrate of Pus3. tRNAGln(UUG) was purified from bulk RNA derived from wild-type and pus3Δ strains, and modified nucleosides were analyzed as described in Materials and Methods. (D) tRNAGln(UUG) is the only Pus3 substrate that substantially improves the growth of a pus3Δ mutant at high temperature. Strains with [2μ LEU2] plasmids expressing tRNA genes as indicated were grown and spotted as in A. The modification status of U38 or U39 is indicated. (E) tRNAGln(UUG) levels in the pus3Δ mutant are not decreased at high temperature. Wild-type and pus3Δ mutants were grown to log phase at 30°C, diluted with prewarmed media at the indicated temperature, and grown for three or four generations as indicated. The cells were harvested and RNA was isolated and analyzed by Northern blot as described in Materials and Methods, with probes as indicated. The levels of tRNAGln(UUG) are indicated below, after normalization to tRNAVal(AAC), which is not a Pus3 substrate, and then normalization to the values of the third generation of wild type at 30°C, itself normalized to tRNAVal(AAC). (F) The pus3Δ mutant does not have an obvious charging defect. Strains were grown as in E at 30°C, 37°C, and 38°C, and then RNA was isolated under acidic conditions and analyzed by acidic Northern blot as described in Materials and Methods. A deacylated control sample (deacyl) was loaded on the left end. Upper and lower arrows denote charged and uncharged tRNA species.

To define the tRNA specificity for suppression of the temperature sensitivity of pus3Δ mutants, we tested each of the 25 tRNA species bearing a Ψ or uncharacterized uridine at residue 38 or 39 in yeast, after overexpression of a representative tRNA gene on a 2μ plasmid containing no other genes. We found that only the plasmid expressing tRNAGln(UUG) substantially improved the growth of the pus3Δ strain at 38°C and 39°C, whereas overproduction of the other tRNA species had no discernable effect on growth (Fig. 1D). As the pus3Δ strain overexpressing tRNAGln(UUG) grew nearly as well as the pus3Δ strain expressing PUS3, these results suggest strongly that tRNAGln(UUG) is the major biologically important target of Pus3 at high temperature.

It was possible that the temperature sensitivity of the pus3Δ mutant was due to reduced levels of tRNAGln(UUG) at high temperature caused by tRNA degradation by the rapid tRNA decay pathway (Chernyakov et al. 2008; Dewe et al. 2012) or the nuclear surveillance pathway (Kadaba et al. 2004), both of which are known to target specific tRNA species in certain hypomodified strains. However, we found that tRNAGln(UUG) levels were unaffected in the pus3Δ mutant after growth at 30°C or after shift to 38°C for three or four generations, relative to the levels of the control tRNAVal(AAC), which is not a substrate for Pus3 and does not have U38 or U39 (Fig. 1E). Furthermore, analysis of tRNAs isolated under acidic conditions to preserve charging showed that charging was unaffected after growth at 37°C or 38°C (Fig. 1F). Thus, the defect in the pus3Δ strain that impairs tRNAGln(UUG) function must be due to some other property of the hypomodified tRNAGln(UUG).

PUS3 has synthetic genetic interactions with genes that introduce mcm5s2U34 in tRNAGln(UUG), tRNAGlu(UUC), and tRNALys(UUU)

Previous high-throughput studies (Costanzo et al. 2010) suggested that PUS3 had multiple negative synthetic genetic interactions with mutations in genes specifying tRNA modifications, including several genes required for formation of mcm5s2U34, which is found on tRNAGln(UUG), tRNAGlu(UUC), and tRNALys(UUU). As tRNAGln(UUG) has mcm5s2U, we reexamined the most severe of these synthetic interactions and determined if reduced function of tRNAGln(UUG) could explain the double-mutant phenotypes. We examined KTI12, which encodes one of several proteins required for formation of the cm5U moiety of the xcm5U34 modifications (Huang et al. 2005), and UBA4, which encodes one of a number of proteins required for synthesis of the s2U moiety of mcm5s2U (Huang et al. 2008). We deleted KTI12 and UBA4 in a pus3Δ [CEN URA3 PUS3] strain, and tested the mutants for growth after introduction of a low copy [CEN LEU2] complementing plasmid, a high copy tQ(UUG) plasmid, control high copy plasmids bearing other tRNAs, or an empty vector, followed by selection against the [CEN URA3 PUS3] plasmid on media containing 5-fluoroorotic acid (5-FOA). We found that kti12Δ pus3Δ mutants were inviable, as the kti12Δ pus3Δ [CEN URA3 PUS3] strain did not grow on media containing 5-FOA when it harbored a [CEN LEU2] empty vector (vec), but behaved like the corresponding single mutant when it harbored a complementing [CEN LEU2 PUS3] or [CEN LEU2 KTI12] plasmid (Fig. 2A).

FIGURE 2.

FIGURE 2.

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PUS3 has negative synthetic genetic interactions with genes required for mcm5s2U modification, which are partially suppressed by tRNAGln(UUG). (A) Overproduction of tRNAGln(UUG) suppresses the synthetic lethality of pus3Δ kti12Δ cells. A pus3Δ kti12Δ[CEN URA3 PUS3] strain was transformed with [LEU2] plasmids as indicated, and cells were grown overnight in SD-Leu medium, serially diluted, and spotted onto SD-Leu medium containing 5-FOA to select against the URA3 plasmid, and plates were incubated at 30°C for 5 d. (lc) CEN plasmid and (hc) 2μ plasmid. (B) Overproduction of tRNAPro(UGG) further improves the growth of the kti12Δ pus3Δ [2μ HIS3 tQ(UUG)] mutant. Strains containing plasmids as indicated were grown in SD-His-Leu medium, diluted, and spotted as in A, and incubated at the indicated temperature for 2 d. (C) The synthetic growth defect of pus3Δ uba4Δcells is suppressed by overproduction of tRNAGln(UUG). A uba4Δ pus3Δ [CEN URA3 PUS3] strain was transformed with [LEU2] plasmids as indicated, and cells were grown and analyzed as in A. (D) Overexpression of other known Uba4 substrate tRNAs does not further improve the growth of the uba4Δ pus3Δ [2μ HIS3 tQ(UUG)] mutant. Strains with plasmids as indicated were grown and analyzed as in B.

Remarkably, overproduction of tRNAGln(UUG) suppressed the lethality of kti12Δ pus3Δ mutants, whereas no suppression was observed by introduction of high copy plasmids overproducing tRNALys(UUU), which has the same mcm5s2U modification as tRNAGln(UUG); tRNAGln(CUG), the other isoacceptor of this tRNA family; or tRNAPro(UGG), another tRNA bearing Ψ38 (Fig. 2A). Overproduction of tRNAGln(UUG) resulted in modest growth at temperatures up to 35°C, and temperature sensitivity at 37°C and higher temperatures (Fig. 2B). We also found that suppression of kti12Δ pus3Δ mutants was further enhanced by overproduction of both tRNAGln(UUG) and tRNAPro(UGG) (which has ncm5U), but was not further enhanced by overproduction of tRNAGln(UUG) in combination with either of the other two tRNAs with mcm5s2U (tRNALys(UUU) or tRNAGlu(UUC)) or any of the other tRNAs containing both xcm5U and encoded U38 or U39 (Fig. 2B).

Similarly, we found that the uba4Δ pus3Δ strain grew poorly and was temperature-sensitive on YPD medium, and that these phenotypes were efficiently suppressed by overexpression of tRNAGln(UUG) (Fig. 2C,D). Overproduction of tRNAGln(UUG) substantially rescued the poor growth of a uba4Δ pus3Δ mutant at all temperatures up to 38°C, but growth was not quite as robust as that of a uba4Δ pus3Δ [CEN LEU2 PUS3] strain (Fig. 2D). Furthermore, suppression was not further enhanced by overproduction of tRNAGln(UUG) in combination with either tRNALys(UUU) or tRNAGlu(UUC), the other tRNAs with mcm5s2U (Fig. 2D).

s2U levels are reduced at 33°C to 39°C in both pus3Δ mutants and wild-type strains

We speculated that the temperature sensitivity of the pus3Δ strain might be due to loss of both Ψ38 and mcm5s2U of tRNAGln(UUG) because of the suppression of the synthetic growth defects of the kti12Δ pus3Δ and the uba4Δ pus3Δ strains by tRNAGln(UUG) overexpression. To test this hypothesis, we purified tRNAGln(UUG) from the pus3Δ strain and the wild-type parent strain BY4741 after growth at 30°C and 37°C, and examined modifications. As expected, the levels of Ψ and m1A were virtually unchanged in both strains at the two different temperatures (Table 2). However, consistent with our hypothesis, we found reduced levels of mcm5s2U in tRNAGln(UUG) from the pus3Δ mutant after growth at 37°C, compared with that at 30°C (0.19 compared with 0.87 mol/mol), accompanied by a commensurate increase in mcm5U levels (from undetectable levels to 0.62 mol/mol) (Table 2; Fig. 3A). This result implies that the temperature-sensitive phenotype of the pus3Δ mutant was due to the lack of Ψ38 in combination with the partial loss of s2U from tRNAGln(UUG) at 37°C.

TABLE 2.

Quantification of nucleosides of tRNAGln(UUG), tRNALys(UUU), and tRNAGlu(UUC) from wild-type and pus3Δ cells after three generations at 30°C and 37°C

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FIGURE 3.

FIGURE 3.

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tRNAGln(UUG) has reduced mcm5s2U levels at 37°C, accompanied by an increase in mcm5U. (A) Loss of the s2U moiety of mcm5s2U from tRNAGln(UUG) occurs at 37°C in the wild-type and the pus3Δ strain. A wild-type strain and a pus3 mutant at log phase at 30°C were harvested three generations after a shift to 37°C, tRNAGln(UUG) was purified, and nucleosides were analyzed as described in Materials and Methods. The HPLC-UV chromatograph of modified nucleosides of tRNAGln(UUG) is shown at 265 nm, and the region containing mcm5s2U and mcm5U modifications is enlarged, with moles of modifications per mol of tRNA indicated next to the corresponding peaks. (B) A schematic of the anticodon loop region of tRNAGln(UUG), tRNALys(UUU), and tRNAGlu(UUC) is shown, all of which bear the mcm5s2U34 modification.

To our surprise, we also found that tRNAGln(UUG) from the wild-type strain had comparably reduced levels of mcm5s2U at 37°C (from 0.85 to 0.20 mol/mol), accompanied by an increase in mcm5U from undetectable levels to 0.69 mol/mol. Furthermore, both wild-type and pus3Δ strains grown at 37°C had comparably reduced levels of s2U in their tRNAGlu(UUC) and tRNALys(UUU) (Fig. 3B), the other two cytoplasmic tRNA species known to have mcm5s2U. For tRNAGlu(UUC) purified from the wild-type strain at 37°C, mcm5s2U levels were reduced from 0.75 to 0.17 mol/mol, while mcm5U increased from undetectable levels to 0.60 mol/mol, whereas other modifications remained at constant levels (Table 2). A similar reduction of mcm5s2U levels and commensurate increase in mcm5U levels was found in tRNAGlu(UUC) from the pus3Δ mutant, and in tRNALys(UUU) from both the wild-type and the pus3Δ strain at 37°C (Table 2). We infer that at 37°C the s2U modification is not efficiently made in the BY4741 wild-type strain and derivative strains. As might be expected, there was no change in the modification levels of representative tRNAs bearing mcm5U34 (tRNAGly(UCC)), ncm5U34 (tRNAPro(UGG)), or ncm5Um34 (tRNALer(UAA)) after growth of either the wild-type or the pus3Δ strain at 37°C (Table 3).

TABLE 3.

Quantification of nucleosides of tRNAGly(UCC), tRNAPro(UGG), and tRNALeu(UAA) from wild-type and pus3Δ cells after three generations at 30°C and 37°C

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Further investigation showed that the reduced levels of the s2U moiety of tRNAs were increasingly obvious as temperatures increased above 30°C. In YPD medium, reduced levels of the s2U moiety of tRNAGln(UUG) were significant after growth for three generations at 33°C (mcm5s2U reduced from 0.92 to 0.78 mol/mol, with a commensurate increase in mcm5U), and were more extreme after growth for three generations at 35°C, 37°C, and 39°C (mcm5s2U levels reduced to 0.64, 0.44, and 0.36 mol/mol, respectively) (Table 4; Fig. 4A). This graded temperature-dependent loss of the s2U from tRNAGln(UUG) suggests increasingly reduced capacity for synthesis of the s2U moiety at elevated temperatures. Consistent with this interpretation, we also found that loss of the s2U moiety occurred gradually as a function of the number of generations after the temperature was increased to 37°C (Table 5; Fig. 4B). The levels of mcm5s2U decreased steadily in each generation, accompanied by an increase in mcm5U, as expected for progressive loss of synthetic capacity for the s2U modification at high temperature.

TABLE 4.

Quantification of nucleosides of tRNAGln(UUG) in BY4741 wild-type cells after three generations at various temperatures

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FIGURE 4.

FIGURE 4.

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The reduced levels of s2U in tRNAGln(UUG) are dependent on temperature and growth medium. (A) Reduced levels of the s2U moiety of tRNAGln(UUG) are detectable after growth for three generations at 33°C and are more extreme at higher temperatures. BY4741 was grown to log phase at 30°C, shifted to different temperatures, and grown for three generations. Then tRNAGln(UUG) was purified and analyzed from harvested cells. (Dark gray bars) mcm5U; (light gray bars) mcm5s2U. (B) Reduced levels of the s2U moiety occur gradually as a function of time after temperature shift to 37°C. BY4741 cells were grown as in A and harvested one to five generations after shifting the temperature to 37°C, and nucleosides in tRNAGln(UUG) were analyzed as in A. (C) Reduced levels of the s2U moiety of tRNAGln(UUG) occur in two widely used wild-type strains in glucose-containing media. Strains were grown in rich medium (YPD), synthetic complete medium (SDC), or YP medium with glycerol (YPG) to log phase and shifted to 37°C as indicated for three generations, followed by purification of tRNAGln(UUG) from harvested cells and nucleoside analysis. (D) Reduced levels of the s2U modification of tRNA are not fully restored within two generations after a shift back to 30°C. BY4741 cells were grown in YPD media for four generations at 37°C as in A, followed by dilution in media prewarmed to 30°C, and growth for two more generations, and tRNAGln(UUG) was purified from cells harvested as indicated and analyzed for nucleosides.

TABLE 5.

Quantification of nucleosides of tRNAGln(UUG) in BY4741 wild-type cells after growth at 30°C and 37°C for various generations

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The reduced s2U levels extended to minimal media and another commonly used strain. We found a similar loss of the s2U moiety of mcm5s2U in tRNAGln(UUG) in the BY4741 strain after growth at 37°C in synthetic complete medium or after growth of the S. cerevisiae W303 strain in YPD medium for three generations at 37°C (Fig. 4C). Thus, the reduced levels of s2U occur in two widely used S. cerevisiae wild-type strains grown at 37°C in conventional media. However, we note that there was a distinctly more modest loss of s2U modification after growth of the BY4741 strain for three generations in YP medium containing glycerol (Fig. 4C).

Interestingly, a shift back to 30°C does not completely correct the defect in s2U modification of tRNA within two generations (Table 6; Fig. 4D). Although there was an increase in mcm5s2U modification in the two generations after the cells were shifted from 37°C back to 30°C, there was only a partial increase. It is striking that the amounts of mcm5U that remain at each generation after the shift back to 30°C are very similar to those predicted if previously matured tRNAGln(UUG) containing mcm5U was not efficiently subsequently modified to mcm5s2U.

TABLE 6.

Quantification of nucleosides of tRNAGln(UUG) in BY4741 wild-type cells after growth at 37°C, followed by shift to 30°C

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Ψ38 affects the function of tRNAGln(UUG) at 30°C

We also found evidence that tRNAGln(UUG) function is compromised by lack of Ψ38 at lower temperature, under conditions where mcm5s2U is intact. Previous experiments established that the normally lethal deletion of the single copy tQ(CUG) gene could be suppressed by overexpression of tRNAGln(UUG), implying that the overexpressed tRNA could decode CAG codons (Johansson et al. 2008). However, we found that this suppression was strongly PUS3-dependent, as the tQ(CUG)Δ [CEN URA3 tQ(CUG)] [2μ LEU2 tQ(UUG)] strain grew well on media containing 5-FOA, but the corresponding tQ(CUG)Δ pus3Δ [CEN URA3 tQ(CUG)] [2μ LEU2 tQ(UUG)] strain did not (Fig. 5).

FIGURE 5.

FIGURE 5.

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The function of tRNAGln(UUG) is compromised by lack of Ψ38 at 30°C. A tQ(CUG)Δ strain and a pus3Δ tQ(CUG)Δ strain harboring either a [CEN URA3 tQ(CUG)] plasmid or a [2μ LEU2 tQ(UUG)] plasmid were grown in YPD, diluted, and spotted on YPD media at the indicated temperatures for 2 d.

At standard temperatures Ψ39 affects the function of tRNATrp(CCA) and of tRNALeu(CAA)am

We examined the synthetic phenotype reported for mutation of PUS3 and TRM10, which encodes the m1G9 methyltransferase (Jackman et al. 2003), because the pus3Δ trm10Δ interaction was the most severe of those reported (Costanzo et al. 2010). We found that pus3Δ trm10Δ [CEN URA3 PUS3] strains were not viable on standard FOA media, but were weakly viable on media containing lower amounts of FOA (Fig. 6A), consistent with previous results demonstrating a growth defect of trm10Δ strains on media containing 5-fluorouracil (Gustavsson and Ronne 2008). This growth defect was fully complemented by a [CEN LEU2 PUS3] plasmid (Fig. 6A) or a [CEN LEU2 TRM10] plasmid (data not shown). We determined which, if any, of the tRNAs that have m1G9 (Swinehart et al. 2013) and Ψ38 or Ψ39 (or an uncharacterized U38 or U39) could improve this growth defect on media containing FOA. We found that tRNATrp(CCA) overexpression suppressed the severe growth defect of the pus3Δ trm10Δ strain, whereas overexpression of the other substrate tRNAs of Trm10 and Pus3 did not; however, the pus3Δ trm10Δ [2μ LEU2 tW(CCA)] strain still grew poorly compared with the control pus3Δ trm10Δ [CEN LEU2 PUS3] strain, particularly at higher temperatures (Fig. 6B). As tRNATrp(CCA) has a relatively unstable anticodon stem compared with other tRNA species (Table 7, −2.4 kcal/mol), this result could be interpreted as the result of improved stability of the anticodon helix by Ψ39, as Ψ has been shown to increase the stability of duplex RNAs (Durant and Davis 1999).

FIGURE 6.

FIGURE 6.

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Ψ39 has an important role in tRNATrp(CCA) and tRNALeu(CAA)am. (A) The severe growth defect of the pus3Δ trm10Δ strain is suppressed by overproduction of tRNATrp(CCA) on FOA medium. Strains containing [LEU2] plasmids as indicated were grown overnight in SD-Leu medium, diluted, and spotted on SD-Leu medium containing half the normal amount of 5-FOA, as trm10Δ mutants were shown to be sensitive to 5-fluorouracil. (B) Overexpression of tRNATrp(CCA) partially suppresses the growth defect of a pus3Δ trm10Δ mutant on rich medium at lower temperatures. Cells growing on FOA medium in A were restreaked on the same FOA medium, and colonies were grown overnight in SD-Leu, spotted on YPD and incubated as indicated temperature for 2 d. (C) The function of tRNALeu(CAA)am, but not tRNATyr(GUA)am or tRNASer(CGA)am, is defective in the absence of Ψ39 at 28°C. Strains containing the integrated RNA-ID reporter expressing GFPam and RFP and an integrated tRNAam suppressor were grown and analyzed for GFP and RFP expression using flow cytometry as described in Materials and Methods. Scatter plots of strains with tY(GUA)am (upper panel); tS(CGA)am (middle panel); and tL(CAA)am (lower panel) are shown for the corresponding wild-type (red) and pus3Δ (blue) strains.

TABLE 7.

Free energy prediction of anticodon stem–loop of yeast cytoplasmic tRNAs, ignoring modifications

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To further investigate the function of Ψ39 in stabilization of the anticodon helix, we compared the function of three tRNAs with Ψ39 and different predicted stabilities of the anticodon stem, by evaluation of nonsense suppression. We examined tRNATyr(GUA), tRNASer(CGA), and tRNALeu(CAA), with predicted anticodon stem–loop stabilities of −2.4, −3.4, and, −4.9 kcal/mol, respectively (Reuter and Mathews 2010), after changing the anticodon to CUA to read the UAG (amber) nonsense codon, and integrating the tRNA into the RNA-ID reporter strain expressing both GFPam and RFP (Dean and Grayhack 2012). Under these conditions, we previously found we could accurately quantify nonsense suppression by using flow cytometry to compare GFP and RFP expression (Guy et al. 2014). We found that suppression in the tL(CAA)am strain was severely reduced in the pus3Δ mutant relative to that in the corresponding PUS3+ strain after growth at 28°C (from GFP/RFP 0.223 to GFP/RFP 0.024), 33°C (from 0.083 to 0.012), or 37°C (from 0.019 to 0.006) (Table 8; Fig. 6C), strongly indicating that Ψ39 has an important role in the function of tRNALeu(CAA). In contrast, the pus3Δ mutation had no effect on suppression in the tY(GUA)am strain relative to that in a PUS3+ strain at all three temperatures (from 0.871 to 0.886 at 28°C, from 0.624 to 0.691 at 33°C, and from 0.351 to 0.380 at 37°C). Furthermore, there was little effect of the pus3Δ mutation on suppression in the tS(CGA)am strains at 28°C (from 0.721 to 0.663), and only a modest reduction of suppression at 33°C and 37°C (from 0.455 to 0.130 and from 0.099 to 0.014, respectively). These results suggest that the role of PUS3 extends to Ψ39, but not in a manner that can be explained by increased stability of the anticodon stem–loop.

TABLE 8.

GFP/RFP values from the RNA-ID reporter in wild-type and pus3Δ strains with tY(GUA)am, tL(CAA)am, or tS(CGA)am suppressors

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DISCUSSION

We have provided evidence here that yeast pus3Δ mutants are temperature-sensitive owing to reduced function of tRNAGln(UUG), because of the absence of Ψ38 in combination with reduced levels of the s2U moiety of mcm5s2U of tRNAGln(UUG). Remarkably, we found that the reduced levels of s2U occur in both the BY4741 strain and the W303 strain at 37°C, two strains that are in widespread use in the yeast community, and that reduced s2U is detectable at temperatures as low as 33°C, and occurs in both YPD and synthetic media. As the loss of s2U is associated with slower decoding of VAA codons and up-regulation of GCN4 by a GCN2-independent mechanism (Zinshteyn and Gilbert 2013), numerous biological effects reported in the literature at even mildly elevated temperatures may need to be reinterpreted because of the accompanying partial loss of the s2U moiety. The more minor reduction of s2U in YP media containing glycerol may be due to the reduced growth rate, which would allow increased time for mcm5s2U biosynthesis, or might be due to different transcription and proteome content in a carbon source requiring respiration (Gasch et al. 2000).

The reduction in the s2U moiety of the mcm5s2U modification at modestly elevated temperatures is part of an emerging theme of altered modification programs observed under different stress conditions. It is intriguing that at 37°C the s2U modification was also previously found to be reduced in yeast mitochondrial tRNALys(UUU), which normally has cmnm5s2U34, whereas the known s2U of mitochondrial tRNAGln(UUG) and tRNAGlu(UUC) was not (Kamenski et al. 2007). It thus seems possible that s2U formation of mitochondrial tRNALys(UUU) has components in common with the cytoplasmic s2U modification that are not shared by the other two mitochondrial tRNA species. Other examples of altered modifications during growth of yeast include increased m5C in the anticodon of tRNALeu(CAA) and loss of m2,2G and Cm during oxidative stress (Chan et al. 2010, 2012), and the increased m5C at C48 and C50 of tRNAHis during the onset of stationary phase, amino acid starvation, and rapamycin treatment (Preston et al. 2013). It thus seems plausible that these alterations in modifications are an integral part of a stress response.

Remarkably, our data suggest that once mature tRNAGln(UUG) is synthesized with mcm5U, the s2U moiety is not easily added to form mcm5s2U, following a return to 30°C and log phase growth. Although this result might imply that there is a biochemical requirement for s2U to be made before the mcm5 moiety is added, available evidence shows instead that several mutants required for mcm5U biosynthesis are also partially lacking the s2U modification (Nakai et al. 2008; Noma et al. 2009). Two other explanations might explain the slow recovery of tRNAs that are fully modified with mcm5s2U. First, once tRNA biogenesis is finished and tRNA with the mcm5U modification enters into the translation cycle, there may be a reduced likelihood for that tRNA to be subsequently modified with the s2U modification. This could occur if the tRNA is being sequestered by the translation machinery and is not able to effectively bind the s2U modification enzymes, or if there is a specific time during tRNA biogenesis when the s2U modification should be made; for example, tRNAPhe maturation requires retrograde transport of spliced tRNA to the nucleus (Murthi et al. 2010), in part to ensure modification of G37 to m1G37 prior to yW formation after reexport (Ohira and Suzuki 2011). Second, there might be limiting biosynthetic capacity for making the s2U modification, thereby requiring a significant amount of time to generate mcm5s2U on all tRNA species after the return to 30°C.

The suppression of the pus3Δ temperature sensitivity by increased dosage of tRNAGln(UUG) emphasizes that, as with other modifications mutants, there is a primary target tRNA for which the modification appears to have the major biological effect (Esberg et al. 2006; Björk et al. 2007; Phizicky and Alfonzo 2010; Dewe et al. 2012; Guy et al. 2012). Two previous reports also emphasize a central role of mcm5s2U of tRNAGln(UUG): the high copy suppression by tRNAGln(UUG) and tRNALys(UUU) of the multiple phenotypes of elp mutants, which lack the cm5U moiety (Esberg et al. 2006); and the enhanced high copy suppression by tRNAGln(UUG) (in addition to tRNALys(UUU)) of the lethality of elp3Δ tuc1Δ mutants, which lack both the cm5U and s2U moieties of mcm5s2U (Björk et al. 2007).

Our results also indicate a distinct role of Ψ38 of tRNAGln(UUG) separate from mcm5s2U, as suppression of the lethality of a tQ(CUG)Δ strain by high copy expression of tRNAGln(UUG) (Johansson et al. 2008) is much more efficient when Pus3 is present than in a pus3Δ mutant; this Pus3 dependence of the tRNAGln(UUG) suppression occurs at 30°C, conditions in which mcm5s2U levels are normal. Consistent with the observation that suppression of the tQ(CUG)Δ lethality by overexpression of tRNAGln(UUG) in a PUS3+ strain requires mcm5s2U (Johansson et al. 2008), we found that suppression was reduced at higher temperatures, when s2U levels are reduced (Fig. 5). The effect of Ψ38 on tRNAGln(UUG) function might be due to reduced ability to decode CAG codons (Johansson et al. 2008), or to an overall reduced functional level of tRNAGln(UUG). Although the precise role of Ψ38 in tRNAGln(UUG) is not clear, Ψ38 is involved in noncanonical interactions with N32 (Auffinger and Westhof 1999), and Ψ stabilizes single-stranded regions (Davis 1995).

Although it is well established that Ψ stabilizes stacking in both single-stranded regions and duplexes of helices by promoting the C3′ endo conformation and coordination of a water molecule (Arnez and Steitz 1994; Davis 1995; Durant and Davis 1999; Charette and Gray 2000), it is not clear that this stabilization is the only significant biological effect of Ψ39. In favor of stabilization is the apparently biased distribution of Ψ39 or an uncharacterized U39 on those yeast cytoplasmic tRNAs with less stable anticodon stems (Table 7), and the observed suppression of the trm10Δ pus3Δ growth defect by the tRNA species with m1G9 and the least stable anticodon stem (tRNATrp(CCA)). However, the significant role for Ψ39 on tRNALeu(CAA)am function and the more modest role for Ψ39 on function of tRNATyr(GUA)am and tRNASer(CGA)am, argues for an additional role of Ψ39 in yeast as tRNALeu(CAA) is predicted to have a much more stable anticodon stem than the two other species. We note that the identity and pairing ability of the 31–39 pair at the bottom of the anticodon stem have both been implicated in first base decoding accuracy of tRNAGln(CUG) (Murray et al. 1998; Kemp et al. 2013), and that Ψ39 has been shown to increase −1 frameshifting in certain constructs (Bekaert and Rousset 2005). Thus, in this case Ψ39 may increase decoding efficiency of tRNALeu(CAA)am. However, its role remains to be defined.

It is intriguing that tRNAGln(UUG) appears to be balanced on a knife edge of function. As reported here, tRNAGln(UUG) function is critically dependent on Pus3 function at all temperatures, and as previously reported, tRNAGln(UUG) and tRNALys(UUU) function in several phenotypes depends on both moieties of mcm5s2U (Esberg et al. 2006; Björk et al. 2007). Thus, tRNAGln(UUG) may have an important regulatory role in the cell. Indeed, it is known that tRNAGln(CUG) function is important for nitrogen sensing (Murray et al. 1998) and it is known that increased tRNAGln(UUG) levels can suppress the nitrogen sensing defect (Kemp et al. 2013) and the lethal phenotype of tQ(CUG)Δ strains (Johansson et al. 2008). As s2U levels appear regulated by temperature in wild-type cells, and yeast mutants lacking either s2U or xcm5U constitutively activate the GCN4 stress response (Zinshteyn and Gilbert 2013), it seems plausible that the stress response at high temperature is in part mediated by the loss of s2U and reduced function of tRNAGln(UUG).

MATERIALS AND METHODS

Yeast strains

Strains used for this study are derived from BY4741 and are listed in Table 9. Because PUS3 (YFL001W) is very near the centromere of chromosome VI, we replaced the first 379 nt of the gene with a bleR marker, similar to the approach previously reported (Carbone et al. 1991; Lecointe et al. 2002), using the pUG66 bleR cassette with forward primer 5′-CCACATGCAATCTTTACTGCCCTACTATAACCTCCCTTGACAGCTGAAGCTTCGTACGC-3′ and reverse primer 5′-CAGATCCACTAGTGGCCTATGCCCATGAATAAGAAGTGTAAACTTGTTCCCTCGATGGTTTTA-3′. We tested eight independent isolates for growth phenotype on different media, all of which were identical, and then transformed this strain with the [CEN URA3 PUS3] plasmid (pFEN011), which contains PUS3 DNA from −452 to +35, ending 16 bp before the outer edge of CEN VI.

TABLE 9.

Strains used in this study

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All double-mutant pus3Δ strains were generated by PCR amplification of DNA from the corresponding KanMX strain in the YKO collection (Open Biosystems), followed by linear transformation of the fragment into a pus3Δ::bleR [CEN URA3 PUS3] strain.

The tQ(CUG)MΔ[CEN URA3 tQ(CUG)] strain was constructed by transformation of BY4741 with the [CEN URA3 tQ(CUG)] plasmid containing the tQ(CUG) in a cassette with the 5′ flanking DNA of tH(GUG)G2, followed by PCR amplification of the hygR marker and linear transformation to delete the tQ(CUG) gene.

The GFPam flow cytometry reporter strain YK613-1 (relevant genotype: BY4741 can1::PGAL1 GFPam PGAL10 RFP) was made by PCR amplification of the PGAL1 GFPam PGAL10 RFP DNA and its adjacent MET15 marker from pEKD1294 (Dean and Grayhack 2012) with CAN1 primers, followed by linear transformation, selection on SD-Met, screening on SD-Arg, and PCR amplification and sequencing. Then we made a pus3Δ derivative strain of the YK613-1 GFPamreporter strain (YLH567-1) by PCR amplification of the pus31-379::bleR cassette from strain YK428-1 and linear transformation. Then tY(GUA)am, tL(CAA)am, and tS(CGA)am derivatives of YK613-1 and YLH567-1 were constructed by linear transformation to integrate the Stu I fragments of plasmids (derived from pAB230-1) containing the corresponding tRNA genes and the HIS3 marker, at the ADE2 locus, resulting in the corresponding ade2::5′tH(GUG)G2::tRNAam::HIS3 strain. Three independent transformants were then tested for each tRNA gene inserted. In addition, three independent versions of strain YLH567-1 were transformed and tested with integrated tY(GUA)am, each yielding the same GFP expression within 1.9%.

Plasmids

Plasmids used in this study are listed in Table 10. Plasmids expressing tRNAs were made either by ligation-independent cloning (LIC) of a tRNA with its own flanking sequence into the 2μ LEU2 plasmid pAVA577 or by insertion of a tRNA sequence into the Bgl II, Xho I site of a tRNA expression plasmid (pMAB813A or pJW097) as previously described (Whipple et al. 2011). The same LIC cloning method was also used to construct plasmids bearing KTI12, UBA4, TRM10, and PUS3. The integrating plasmids for the wild-type tRNAs and tRNA variants were constructed by replacement of FLuc DNA with the corresponding tRNA sequence in the Bgl II, Xho I site of plasmid pAB230-1, essentially the same as described previously (Guy et al. 2014). The integrating GFP–RFP reporter plasmid pEKD1294 was derived from pEKD1024 (Dean and Grayhack 2012) by LIC cloning to insert a UAG amber stop codon at amino acid 7 of GFP, using the forward oligo 5′-AATTCCATCAACCTTAATAAAATGTCTACTGAAGTTCAATAGCAAAACGCATCCACCA-3′ and reverse oligo 5′-CTTCCAAACCACTGGTGGATGCGTTTTGCTATTGAACTTCAGTAGACATTTT-3′.

TABLE 10.

Plasmids used in this study

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Growth of yeast strains

Wild-type and pus3Δ strains were grown overnight in YPD or other medium at 30°C, inoculated into fresh medium for one generation, and then diluted as necessary into prewarmed media at the desired temperature, and grown at that temperature in a shaking water bath for subsequent generations as noted, prior to cell harvest and quick freezing of pellets.

Northern blot analysis

Bulk RNA was prepared from ∼3 OD pellets using glass beads, and ∼1 μg RNA was resolved by PAGE and analyzed as previously described (Alexandrov et al. 2006). For analysis of charging, RNA was prepared under acidic conditions (pH 4.5) and resolved on 6.5% acrylamide gels at pH 5 for 15 h at 4°C as described (Alexandrov et al. 2006).

Extraction of bulk low molecular-weight RNA from yeast and purification of tRNA

Bulk RNA was extracted from ∼300 OD pellets using hot phenol, and tRNA was purified from 1.25 mg bulk RNA using 5-biotinylated oligonucleotides (Integrated DNA Technologies) complementary to the corresponding tRNA sequences, as described (Jackman et al. 2003).

HPLC analysis of nucleosides from tRNA

Purified tRNA (1.25 μg) was digested with 0.5 μg P1 nuclease, followed by 1 unit of calf intestinal alkaline phosphatase, and nucleosides were subjected to HPLC and quantified based on extinction coefficients, as described (Jackman et al. 2003), using parameters for mcm5U (Gray 1976) and mcm5s2U (Baczynskyj et al. 1969).

Flow cytometry

Strains were grown for 24 h at 28°C in S-His dropout medium containing 2% raffinose, followed by overnight growth in YP medium containing 2% galactose and 2% raffinose supplemented with 80 mg/L adenine, dilution and growth to OD ∼1, followed by flow cytometry as described (Guy et al. 2014), and data analysis using FlowJo software. Cells that passed the RFP cutoff of 5 × 103 were used to determine a median GFP and median RFP for that sample and a calculated GFP/RFP, and biological triplicates were used to obtain an overall median GFP/RFP and a standard deviation.

ACKNOWLEDGMENTS

We thank Elizabeth Grayhack for valuable discussions and comments during the course of this work and Michael Guy for comments on the manuscript. This research was supported by National Institutes of Health (NIH) grant GM052347 to E.M.P.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.048173.114.

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