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. 2011 Jun;17(6):1100–1110. doi: 10.1261/rna.2652611

A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

Sonia D'Silva 1, Steffen J Haider 1, Eric M Phizicky 1
PMCID: PMC3096042  PMID: 21518804

Abstract

The 3-methylcytidine (m3C) modification is widely found in eukaryotic species of tRNASer, tRNAThr, and tRNAArg; at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNASer. Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNAThr(IGU) from abp140-Δ strains lacks m3C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m3C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m3C and since quantitative analysis shows explicitly that tRNAThr(IGU) from an abp140-Δ strain lacks m3C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m3C methyltransferase activity, which is specific for tRNAThr(IGU) and not tRNAPhe and occurs specifically at C32. We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m3C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m2,2G26) are sensitive to low concentrations of cycloheximide.

Keywords: 3-methycytidine, S. cerevisiae, TRM140, ABP140, tRNA, frameshift

INTRODUCTION

tRNA molecules are widely modified in nature, with a total of 92 chemically distinct tRNA modifications identified in different organisms and entered in the RNA modification database (http://rna-mdb.cas.albany.edu/RNAmods/). In the yeast Saccharomyces cerevisiae, 25 different tRNA modifications are found, at 36 different positions, among the 34 sequenced tRNA species (Sprinzl and Vassilenko 2005), with an average of 12.6 modifications per tRNA species (Phizicky and Hopper 2010). These modifications display two broadly defined roles. First, a number of modifications in and around the anti-codon loop play distinct roles in translation, affecting charging of the tRNA with the correct amino acid, reading frame maintenance, or the efficiency and/or accuracy of translation (Phizicky and Hopper 2010). Second, a number of modifications in the main body of the tRNA remote from the anti-codon have a role in the biogenesis or maintenance of tRNA, and mutations in these modifications lead to turnover of pre-tRNA or mature tRNA by different quality control pathways (Kadaba et al. 2004; Chernyakov et al. 2008). Over the past decade, the enzymes and their cognate genes responsible for most of these modifications have been defined (Phizicky and Hopper 2010).

One of the remaining unidentified yeast genes encoding modification enzymes is the methyltransferase responsible for formation of 3-methylcytidine (m3C). The m3C modification is widely found in eukaryotic tRNASer, tRNAThr, and tRNAArg tRNA species at residue 32 of the anti-codon loop (Sprinzl and Vassilenko 2005). m3C32 is found in all four characterized cytoplasmic tRNAThr species from animals or fungi, in bovine liver mitochondrial tRNAThr(UGU), in all but one of the 15 characterized cytoplasmic tRNASer species from plants and animals, in four of the five characterized fungal cytoplasmic tRNASer species with an encoded C32 residue, and in bovine mitochondrial tRNASer(UGA), as well as in two of the five characterized tRNAArg species from animals. Curiously, m3C is also found at residue e2 of the variable loop of the seven cytoplasmic tRNASer species from animals. In the yeast S. cerevisiae, m3C is found at C32 of tRNAThr(IGU), tRNASer(UGA), and tRNASer(CGA) (Weissenbach et al. 1977; Sprinzl and Vassilenko 2005; Dunin-Horkawicz et al. 2006).

It seems likely that the m3C32 modification would have an important role in the cell, since residue 32 of the anti-codon loop interacts with residue 38 to maintain the structure of the anti-codon loop (Auffinger and Westhof 1999, 2001), an interaction that is known to be important for decoding accuracy (Olejniczak et al. 2005; Olejniczak and Uhlenbeck 2006; Ledoux et al. 2009). However, since its discovery some 47 yr ago (Hall 1963), little has been discovered about the biology and function of m3C or about the rules that govern the specificity of this modification in vivo.

To begin to define the biochemical basis of m3C modification in the cell and the roles of the modification, we have carried out a systematic screen to identify the gene encoding the m3C methyltransferase responsible for this modification. To our surprise, we find that the methyltransferase is the actin-binding protein Abp140p (Asakura et al. 1998), which is necessary and sufficient for the m3C modification (now renamed TRM140 to indicate its tRNA modification activity). It is intriguing that ABP140 is translated by a programed frameshift (Farabaugh et al. 2006b), which fuses an upstream ORF that is variable among different species and implicated in organization of the actin cytoskeleton in the cell (Asakura et al. 1998), with a downstream ORF that, like m3C, is conserved among eukaryotes. We find that this downstream ORF, which has a SAM binding domain (Katz et al. 2003), is necessary and sufficient for m3C methyltransferase activity in vitro and in vivo. We also find that although abp140-Δ strains have no obvious growth phenotype, abp140-Δ trm1-Δ cells, which are missing both m3C and m2,2G, have a distinct, but modest, growth defect in the presence of a translation inhibitor, suggesting a role for m3C in translation.

RESULTS

The yeast ABP140 gene is required for formation of m3C

To determine the gene responsible for m3C modification of tRNA in yeast, we first developed a sensitive primer extension assay to detect the modification, using yeast tRNAThr(IGU), which is known to contain m3C32 (Weissenbach et al. 1977). We purified tRNAThr(IGU) from wild-type yeast cells, annealed a labeled primer designed to pair with the tRNA from residue 55 in the T-loop through residue 35 in the anti-codon loop (Fig. 1A), and extended the primer with reverse transcriptase. Extension resulted in a product that terminated at residue 33 because of the presence of the m3C modification at C32 (Fig. 1B, lanes a–c). In contrast, primer extension of the same tRNA after treatment with the bacterial demethylase AlkB to eliminate the m3C residue (Aas et al. 2003), as well as the m2,2G modification, resulted in a fully extended primer extension product (Fig. 1B, lanes d, e). Thus, the presence and absence of the m3C residue could be scored based on the length of the primer extension product.

FIGURE 1.

FIGURE 1.

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Identification of the gene responsible for m3C modification of yeast tRNAThr(IGU). (A) Schematic of tRNAThr(IGU) and primer used for assay of m3C32. Modifications found on tRNAThr(IGU) are indicated (Weissenbach et al. 1977). The oligonucleotide used for primer extension by reverse transcriptase anneals to residues 55–35. Primer extension of tRNAThr(IGU) species that contain m3C32 will terminate at residue 33, whereas species lacking m3C32 will terminate at residue 27 before m2,2G26. m2G indicates N2-dimethylguanosine; D, dihydrouridine; m2,2G, N2, N2-dimethylguanosine; I, inosine; t6A, N6-threonylcarbamoyladenosine; Ψ, pseudouridine; m5C, 5-methylcytidine; T, ribothymidine; and m1A, 1-methyladenosine. (B) Validation of primer extension assay with AlkB-treated tRNAThr(IGU). tRNAThr(IGU) purified from wild-type yeast cells was treated with the bacterial demethylating enzyme AlkB, and the resultant product was analyzed by the primer extension assay, as described in Materials and Methods. B indicates bulk RNA; P, purified tRNA; FL, full-length; a, 2 μg untreated bulk RNA; b, c, untreated tRNAThr(IGU) (0.02 μg and 0.2 μg); d, e, AlkB-treated tRNAThr(IGU) (0.02 μg and 0.2 μg); and f, no input RNA. (C) An abp140-Δ strain lacks the m3C primer extension block. Bulk RNA from a set of candidate deletion strains was screened by primer extension. RNA from the abp140-Δ strain lacking ORF YOR239W (lane 15) lacks the m3C primer extension block and has a block corresponding to m2,2G26. WT indicates RNA from wild-type control strain; AlkB, AlkB treated tRNAThr(IGU); and −, no RNA.

To identify the gene responsible for m3C modification of tRNAThr(IGU), we employed the primer extension assay to screen candidates from the yeast haploid deletion collection. Based on a text search for methyltransferases in SGD (http://www.yeastgenome.org/cgi-bin/search/textSearch.pl?query=methyltransferase&type=headline), a search of Gcd14-like domains in SGD (http://www.yeastgenome.org/cgi-bin/protein/domainPage.pl?dbid=S000003661), and a methyltransferase search in the Modomics database (Dunin-Horkawicz et al. 2006), we came up with a list of 86 strains with deletions of nonessential genes that encode known methyltransferases, uncharacterized ORFs with similarity to known methyltransferases, or known partners of methyltransferase proteins (Supplemental Table S1). RNA prepared from 77 of these strains was analyzed, yielding one candidate with an extended primer extension product (Fig. 1C). This strain has a deletion of ABP140, which encodes an actin binding protein that co-localizes with actin cables and cortical actin patches (Asakura et al. 1998) and which is synthesized as a programed frameshift of ORF YOR239W with YOR240W (Farabaugh et al. 2006b).

To confirm that ABP140 is responsible for the primer extension block indicative of m3C modification of tRNAThr(IGU), we reconstructed and tested abp140 deletion strains. As anticipated, we find that the primer extension block is absent in strains lacking either one or both ORFs comprising ABP140 (Fig. 2A) and is restored in the deletion strains by introduction of a CEN vector bearing wild-type ABP140, but not by the empty vector (Fig. 2A).

FIGURE 2.

FIGURE 2.

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ABP140 is required for m3C modification of tRNAThr and tRNASer. (A) Lack of the m3C primer extension block is due to lack of ABP140. ORFs YOR239W and/or YOR240W were deleted by transformation of a wild-type strain (BY4741), and 2 μg RNA from these strains was assayed for the m3C primer extension block after transformation with a CEN ABP140 plasmid (lanes a,c,e), or with an empty vector (lanes b,d,f), as indicated. (Lane g) No RNA. Note that the m3C and m2,2G arrows point to the residue immediately preceding the modified residue that causes the primer extension block. (B) ABP140 is required for m3C modification of other tRNAThr species. Bulk RNA (2 μg) from a wild-type strain (lanes b,e,h) and an abp140-Δ strain (a,d,g) was assayed by primer extension using 5′-labeled primers designed against tRNAThr(IGU) (a–c), tRNAThr(UGU) (d–f), and tRNAThr(CGU) (g–i). (Lanes c,f,i) no RNA controls; *, unexplained primer extension stop or pause in tRNAThr(CGU). (C) ABP140 is required for m3C modification of tRNASer(CGA), tRNASer(UGA) and tRNASer(GCU). Bulk RNA (2 μg) from a wild-type strain (lanes a,d) and an abp140-Δ strain (b,e) was assayed by primer extension using 5′-labeled primers designed against tRNASer(CGA) (a–c), tRNASer(UGA) (d–f), and tRNASer(GCU) (g–i). (D) HPLC analysis of tRNAThr(IGU) purified from an abp140-Δ (trm140-Δ) strain. tRNAThr(IGU) prepared from wild-type and abp140-Δ strains was digested to nucleosides and chromatographed on an HPLC column, as described in Materials and Methods.

To determine if other tRNAThr and tRNASer species are also substrates for the presumed m3C modification directed by ABP140, we used a similar primer extension assay. Both tRNAThr(UGU) and tRNAThr(CGU) from wild-type cells also have the primer extension block expected of m3C modification at residue 32 (Fig. 2B, lanes e, h), but this primer extension block is absent in RNA from abp140Δ strains (lanes d, g). Similarly tRNASer(CGA), tRNASer(UGA), and tRNASer(GCU) each have the primer extension block expected from m3C modification of the tRNA in wild-type cells, but the primer extension block is not observed in abp140-Δ cells (Fig. 2C).

To confirm the interpretation of our primer extension results that the primer extension block at residue 33 is due to m3C modification at residue 32, we directly measured m3C levels in tRNAThr(IGU). We purified tRNAThr(IGU) from log phase cultures of the wild-type and deletion strains lacking either YOR239W or YOR240W, digested the RNA to nucleosides, and analyzed the nucleoside content by reverse-phase HPLC chromatography, as described in the Materials and Methods. We find that tRNAThr(IGU) from wild-type cells has approximately the expected levels of all nucleosides, including 0.92 mol of m3C, whereas tRNAThr(IGU) from strains lacking either YOR239W or YOR240W has no detectable m3C but has otherwise similar levels of other nucleosides (Fig. 2D). These observations demonstrate directly that ABP140 is required for the m3C modification of tRNAThr(IGU) in yeast, and therefore we infer that ABP140 is required for m3C32 formation for all six tRNAThr and tRNASer species for which m3C is documented.

Based on these results, and the results below, we refer to the gene ABP140 by the name TRM140, in keeping with nomenclature for other tRNA methyltransferases.

Trm140p (Abp140p) is sufficient for m3C modification of tRNAThr(IGU) in vitro

To determine if Trm140p (Abp140p) is required for m3C modification of tRNAThr(IGU) in vitro, we prepared crude extracts from the wild-type and trm140-Δ (abp140-Δ) strains and assayed their m3C methyltransferase activity with S-adenosylmethionine (SAM), using tRNAThr(IGU) transcribed in vitro with [α-32P] CTP. After incubation of the transcripts with extracts, we digested the tRNA with P1 nuclease to generate 5′-[32P]-labeled CMP and modified CMP residues, and resolved the products by thin-layer chromatography (TLC). We find that crude extracts from wild-type yeast cells have m3C methyltransferase activity (Fig. 3A, lanes a–d), since a modified CMP product is detected after resolution of the nucleotide reaction products on a PEI-cellulose TLC plate. This product co-migrates with an authentic labeled m3C spot, made by reaction of [α-32P] CTP-labeled transcript with DMS, followed by P1 nuclease treatment, which is known to generate 5′-[32P]m3C (p*m3C) (Heyman et al. 1994) This p*m3C product is not detected in an extract from the trm140-Δ (abp140-Δ) strain, even after overexposure of the TLC plate (Fig. 3A, lanes e–h), demonstrating that m3C methyltransferase activity in extracts requires Trm140p (Abp140p). Moreover, extracts prepared from cells overexpressing full-length Trm140p with a nucleotide deleted to correct the need for a programed frameshift yielded about ninefold more m3C methyltransferase activity, based on the titration (Fig. 3A, lanes i–l), demonstrating that Trm140p is the limiting component of methyltransferase activity in extracts. Furthermore, the m3C methyltransferase activity is specific for its tRNA substrate, since no p*m3C spot is detected after assay of [α-32P] CTP-labeled tRNAPhe transcript with either wild-type extracts or extracts from cells overexpressing Trm140p (Fig. 3A, lanes m, o), although tRNAPhe has a C32 residue and although the tRNAPhe is clearly able to be modified by another modification activity that is present in the extracts.

FIGURE 3.

FIGURE 3.

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Trm140p (Abp140p) is sufficient for m3C methyltransferase activity. (A) Trm140p is the limiting component for detection of m3C methyltransferase activity in yeast extracts. tRNAThr(IGU) transcribed in vitro with [α-32P] CTP was incubated in methyltransferase buffer containing serial threefold dilutions of extract from wild-type cells (lanes a–c), from trm140-Δ cells (lanes e–g), or from wild-type cells overexpressing frame-fixed Trm140p (Trm140-ff) in which the reading frames of ORFs YOR239W and YOR240W were deliberately fused (lanes i–k) for 2 h at 30°C; then RNA was digested with P1 nuclease; and products were analyzed by chromatography on a PEI-cellulose developed in 0.25 M lithium chloride, as described in Materials and Methods. (m–o) Methyltransferase activity assayed with [α-32P] CTP-transcribed tRNAPhe with the highest concentration of extracts from wild-type cells (m), trm140-Δ cells (n), or wild-type cells overexpressing Trm140p (o). (Lane q) p*m3C control, generated by DMS-treatment of an [α-32P] CTP-labeled tRNAThr(IGU) transcript; (lane r) P*i control. (B) Trm140p expressed and purified from yeast or from E. coli has m3C methyltransferase activity. Methyltransferase activity was examined as described in A, with [α-32P] CTP-transcribed tRNAThr(IGU), using preparations of frame-fixed Trm140p expressed and purified from yeast (lanes a,b), frame-fixed Trm140p expressed and purified from E. coli (d,e), ORF YOR240W (the C-terminal SAM domain) expressed and purified from E. coli (f,g), or with equivalent volumes of a parallel mock purification from E. coli cells containing an empty vector (h,i). c,j, no protein; k–s, assay of methyltransferase using [α-32P] CTP-transcribed tRNAPhe; t, DMS control.

To determine if Trm140p is sufficient for m3C methyltransferase activity, we expressed the gene with a C-terminal affinity tag in yeast or an N-terminal affinity tag in Escherichia coli and assayed activity after affinity purification (see Materials and Methods). To maximize production of the full-length protein, we first deleted a nucleotide at the junction between ORF YOR239W and ORF YOR240W to encode the full-length fusion protein (labeled ff in the figures to indicate frame-fixed), but surprisingly, immunoblot analysis using antibody against the C-terminal tag shows that the expression of the frame-fixed Trm140p in yeast is very similar to that in a parallel construct with the programed frameshift (Supplemental Fig. S1). We find that purified Trm140-ff protein (Supplemental Fig. S2A) exhibits readily detectable m3C methyltransferase activity in vitro (Fig. 3B, lanes a, b), and activity of the protein is very similar whether derived from a frame-fixed construct or the native construct (Supplemental Fig. S3, cf. lanes a–d and e–h), whereas the mock purification control from yeast with an empty vector has no detectable activity (Supplemental Fig. S3, lanes i–m). Activity is more prominent at 0 or 0.1 mM MgCl2 and is significantly reduced at 1 mM MgCl2 and at 10 mM MgCl2 (Supplemental Fig. S3). As is true in yeast crude extracts, Trm140p purified from yeast is specific, since activity is not detected with tRNAPhe (Fig. 3B, lanes k, l).

Both m3C methyltransferase activity and specificity are maintained after expression in E. coli, which does not normally have the modification or the activity. The frame-fixed Trm140p purified after expression in E. coli (Supplemental Fig. S2B) has readily detectable m3C methyltransferase activity on tRNAThr (Fig. 3B, lanes d, e), but not on tRNAPhe (lanes n, o), whereas no activity is detected in the mock purification from the corresponding E. coli vector control strain (lanes h, i). This finding demonstrates clearly that Trm140p alone is sufficient for m3C methyltransferase activity and for tRNA specificity in the absence of other yeast proteins, although we have not yet quantitatively compared levels of activity from the protein produced in yeast and E. coli.

Trm140p modifies tRNAThr(IGU) at position 32

Two lines of evidence suggest that Trm140p catalyzes m3C modification at C32 of tRNAThr(IGU), rather than at another position. First, formation of m3C requires the presence of the C32 residue of tRNAThr(IGU). Thus, whereas Trm140p readily catalyzes formation of m3C on a wild-type transcript of tRNAThr(IGU), Trm140p does not have detectable activity on a tRNAThr(IGU) transcript with a C32A mutation (Fig. 4A, cf. lanes a, b). This result strongly suggests that C32 is the target for methylation, although it is possible that the C32A mutation instead alters recognition of the site of modification. Second, primer extension of in vitro modified tRNAThr(IGU) demonstrates that the modification occurs at residue C32. To this end, we demonstrated that Trm140 activity was not saturated at concentrations of tRNA up to 800 nM, by competition experiments with labeled transcript and purified unlabeled tRNAThr(IGU) (data not shown). In parallel, we treated 800 nM tRNAThr(IGU) from a trm140-Δ strain with the same amount of Trm140p (or with buffer) in the absence of labeled transcript. The treated products were then subjected to primer extension with the probe for tRNAThr(IGU). We find that tRNAThr(IGU) treated with the Trm140p protein generated the m3C primer extension block at residue 33 (Fig. 4B, lane b), while tRNAThr(IGU) treated only with buffer did not have the m3C primer-extension block (lane c). This result demonstrates that Trm140p modifies C32 of tRNAThr(IGU). Furthermore, since Trm140p modifies only ≤1 mol of cytidine nucleotide per mole of tRNAThr(IGU), its specificity is limited to residue C32.

FIGURE 4.

FIGURE 4.

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Trm140p modifies tRNAThr(IGU) at position 32. (A) Trm140p does not methylate a C32A variant of tRNAThr(IGU). Trm140-ff protein purified from yeast (0.13 μg/μL) was assayed for m3C methyltransferase activity with [α-32P] CTP-transcribed tRNAThr(IGU) (a), tRNAThr(IGU) C32A variant (b), or tRNAPhe (c) for 24 h at 30°C and analyzed as described in Figure 3A. (Lanes d–f) tRNAs treated with buffer controls; (lanes g,h) untreated (g) or DMS-treated (h) tRNAPhe(GAA), used as p*m3C control; (lane i) Pi control. (B) Modification of unlabeled purified tRNAThr(IGU) by Trm140p leads to an m3C32 primer extension block; 0.8 μg tRNAThr(IGU) purified from wild-type cells (lane a) or trm140-Δ strains (lanes b,c) was incubated for 24 h at 30°C in methyltransferase assay buffer with 0.13 μg/μL Trm140-ff protein purified from yeast (lanes a,b) or with buffer control (lane c), and the location of the m3C modification was analyzed by primer extension, as described in Figure 2. (Lanes e,f) primer extension of 0.8 μg untreated tRNAThr(IGU) from trm140-Δ (e) and wild-type (f) strains; (lane d) no RNA.

ORF YOR239W is not required for the methyltransferase activity of Trm140p

Since Trm140p is synthesized by a programed frameshift to fuse ORFs YOR239W and YOR240W, it seems plausible that both ORFs are required for activity. However, prior experiments suggest that YOR239W encodes the protein moiety that interacts with actin (Gao and Bretscher 2008; Riedl et al. 2008), and evolutionary analysis of Abp140p demonstrates that this ORF is poorly conserved, whereas YOR240W, which encodes the methyltransferase domain, is widely conserved among eukaryotes (Asakura et al. 1998; Katz et al. 2003; Farabaugh et al. 2006a). Since m3C is also widely conserved among eukaryotes, this analysis suggests that m3C modification activity might be encoded solely by YOR240W, although it is also possible that the conserved programed frameshift that fuses the methyltransferase domain and the actin binding domain is catalytically or biologically important within S. cerevisiae and related organisms (Farabaugh et al. 2006a).

To address the link between the two domains of Trm140p, we evaluated the m3C methyltransferase activity of the ORF YOR240W protein in vitro and in vivo. We cloned ORF YOR240W (comprising residues 277-628) with an N-terminal ATG start codon into an E. coli expression vector, expressed and purified the protein (Supplemental Fig. S2), and then assayed activity. We find that purified His6-ORF240 has efficient m3C methyltransferase activity (Fig. 3B, lanes f, g), and that this activity is specific for tRNAThr (lanes p, q). Thus, the SAM domain encoded by ORF YOR240W (Katz et al. 2003) is sufficient for the m3C methyltransferase activity in vitro, and the N-terminal domain comprising ORF YOR239W is dispensable for both activity and specificity in vitro.

To determine if ORF YOR240W is sufficient for function in vivo, we deleted the N-terminal domain of TRM140 comprising ORF YOR239W from amino acids 12–276 and expressed TRM140-Δ12-276 in a CEN plasmid. The resulting plasmid (CEN ORF TRM140-Δ12-276) was introduced into a trm140-Δ strain, and the m3C modification status of tRNAThr(IGU) was analyzed by primer extension assay of the RNA. We find that the primer extension block diagnostic of m3C modification is observed when either the CEN TRM140 plasmid or the CEN ORF TRM140-Δ12-276 plasmid is present (Fig. 5, lanes a, b), but no primer extension product is observed when the empty vector is expressed (lane c). These results demonstrate in vivo that the m3C methyltransferase activity of Trm140p on tRNAThr(IGU) is independent of ORF YOR239W, although Trm140-Δ12-276 protein might still bind actin, since the N-terminal 17 amino acids of Trm140p (Abp140p) have been ascribed to this function (Riedl et al. 2008).

FIGURE 5.

FIGURE 5.

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ORF YOR239W is not required for m3C methyltransferase activity in vivo. A trm140-Δ strain was transformed with CEN plasmids containing TRM140-Δ12-276 (lane a), TRM140 (lane b), or an empty vector (lane c), and bulk RNA (2 μg) prepared from these strains was subjected to primer extension as described in Figure 2. (Lane d) No RNA.

trm140-Δ trm1-Δ strains have a mild growth defect in the presence of cycloheximide

Surprisingly, we find that trm140-Δ strains have no obvious growth defects under a variety of standard growth conditions. Thus, when assayed by standard serial dilution spotting experiments, we were unable to detect any growth defect on rich media (YP) or minimal synthetic media (S), with either glucose (D) or glycerol (G) as carbon source, using a temperature range from 18°C–38°C. In addition, the trm140-Δ strain is not sensitive to drugs such as cycloheximide, paramomycin, puromycin, and hygromycin (data not shown). The lack of obvious phenotype of a trm140-Δ strain is somewhat surprising because of the location of the m3C modification in the anti-codon loop at residue 32 (see also Discussion). However, we find that a trm140-Δ trm1-Δ strain is mildly sensitive to low concentrations of cycloheximide (0.25–0.3 μg/mL) on rich (YPD) media (Fig. 6A) and that this sensitivity is complemented by reintroduction of a CEN TRM140 or a CEN TRM1 plasmid, but not by an empty vector control (Fig. 6B). This mild phenotype in the presence of cycloheximide suggests that lack of the m3C modification does impair translation, albeit mildly.

FIGURE 6.

FIGURE 6.

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A trm140-Δ trm1Δ mutant is sensitive to cycloheximide. (A) Enhanced cycloheximide sensitivity is observed in a trm140-Δ trm1-Δ strain. Wild-type (BY4741), abp140-Δ (ySD179), trm1-Δ (yLN005), and abp140-Δ trm1-Δ (ySD576) strains were grown at 30°C overnight, and 0.3 OD cells were serially diluted by 10-fold, spotted on rich (YPD) medium containing either 0, 0.1, or 0.3 μg/mL cycloheximide, and incubated at 30°C for 2 d (without cycloheximide) or 3 d (with cycloheximide). (B) Cycloheximide growth inhibition of a trm140-Δ trm1Δ mutant is rescued by expression of TRM140 or TRM1. trm140-Δ trm1-Δ cells (ySD576) were transformed with a LEU2 CEN plasmid containing TRM140 (pSD158) or TRM1 (pMG67A) or with the control empty CEN LEU2 vector (AVA581), and transformants were grown overnight at 30°C in SD-Leu medium and spotted on YPD, YPG (glycerol) or YPD containing 0.25 μg/mL cycloheximide. Control wild-type (BY4741), trm140-Δ (ySD179), and trm1-Δ (yLN005) strains were transformed with the same empty vector and tested in parallel. Plates were incubated at 30°C for 2–3 d.

DISCUSSION

We have provided evidence that the S. cerevisiae gene ABP140 (now called TRM140) is necessary and sufficient for m3C modification at C32 of known tRNAs with this modification. TRM140 is required for m3C modification, since the primer extension block normally imposed by m3C is absent in trm140-Δ mutants in all six tRNA species known to have this modification and since direct examination of nucleosides shows that purified tRNAThr(IGU) from a trm140-Δ strain specifically lacks m3C that is normally found when the tRNA is purified from wild-type cells. We have also shown directly that Trm140p has m3C methyltransferase activity on tRNAThr(IGU) by an assay of protein purified after expression in yeast or E. coli. Since this activity is specific for tRNAThr compared with tRNAPhe and occurs specifically at residue 32, we conclude that Trm140p is sufficient for m3C methyltransferase activity, although we have not quantitatively compared the activities, or examined other substrates in vitro.

It is intriguing that Trm140p is synthesized by a programed +1 frameshift in which ORF YOR239W is fused to ORF YOR240W (Asakura et al. 1998), resulting in the fusion of an actin binding domain with the methyltransferase. Farabaugh and colleagues have done an extensive evolutionary analysis of the TRM140 (ABP140) gene and its programed frameshift, and find that ORF YOR239W is poorly conserved, whereas ORF YOR240W encodes a highly conserved SAM-methyltransferase domain (Katz et al. 2003; Farabaugh et al. 2006b). Furthermore, they find that the frameshift has been evolutionarily conserved for 150 Myr, since it is extensively found in the budding yeasts, but not in other fungi (Farabaugh et al. 2006b).

There is good evidence that both domains of this protein are functional in yeast but in apparently different biological processes. Although most of the N-terminal ORF YOR239W is very poorly conserved, it is clearly implicated in binding to actin in S. cerevisiae, since Abp140p was originally identified as an F-actin binding protein that co-localized with cortical actin patches and cytoplasmic actin cables (Asakura et al. 1998), and since the N-terminal 17 amino acids are sufficient for this activity (Riedl et al. 2008). Furthermore, the N-terminal ORF has biological function, since overexpression of the fragment encoding residues 1–218 of YOR239W suppresses the lethality conferred by overexpression of a domain of the formin Bni1p (Gao and Bretscher 2008), which nucleates actin filament formation (Pruyne et al. 2002).

Our finding that the C-terminal ORF YOR240W is necessary and sufficient for m3C methyltransferase activity in vitro and in vivo is consistent with the high conservation of both the ORF and m3C in fungi, metazoans, and plants and the lack of conservation of either ORF YOR239W or the frameshift (Farabaugh et al. 2006b), and suggests that the homologous ORFs encode the m3C-methyltransferase activity. Thus, it appears that Trm140 protein fortuitously combines two ORFs with unrelated function, much like PUS8/RIB2 encodes the pseudouridyase that modifies position 32 in cytoplasmic tRNAs, as well as the unrelated DRAP (2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5′-phosphate) deaminase that acts in riboflavin biosynthesis (Behm-Ansmant et al. 2004), although it remains to be explicitly determined if actin binding is important in vivo for Trm140p function.

The finding that trm140-Δ mutants have no detectable growth phenotype under a variety of conditions is somewhat surprising, given the widespread conservation of the m3C32 modification, the documented importance of the anti-codon loop in translation, the known interaction of residue 32 with residue 38 to affect structure of the anti-codon loop (Auffinger and Westhof 1999, 2001), and the altered ribosome binding and translational fidelity of variants in these residues (Olejniczak et al. 2005; Olejniczak and Uhlenbeck 2006; Ledoux et al. 2009). We note, however, that there are large differences in the severity of growth defects observed in strains with mutations that prevent modification of residues in the anti-codon loop. Thus, severe growth defects are associated with loss of I34, m1G37, and t6A37 (Gerber and Keller 1999; Bjork et al. 2001; El Yacoubi et al. 2009); less severe but pronounced growth defects are associated with loss of Cm32 and Nm34, and Ψ38 and Ψ39 (Lecointe et al. 1998, 2002; Pintard et al. 2002); and milder but distinct translational differences are associated with loss of mcm5s2U34, yW37, and i6A37 (Esberg et al. 2006; Waas et al. 2007). The lack of observable phenotype of cells lacking m3C32 is similar to that observed in mutants lacking I37 (Gerber et al. 1998), or the pseudouridylase moiety of PUS8, which is responsible for Ψ32 modification (Behm-Ansmant et al. 2004). Modification of residue 32 may itself confer only subtle effects on translation, based on the lack of obvious phenotype of trm140-Δ mutants and of pus8 alleles that specifically affect pseudouridylation, although the pronounced growth defect of trm7-Δ mutants might be partly due to its modification of residue 32 as well as residue 34 (Pintard et al. 2002).

Indeed, our evidence suggests that m3C modification has a distinct, albeit subtle translation effect, since a trm140-Δ trm1-Δ strain is mildly sensitive to low concentrations of cycloheximide, and this sensitivity is complemented by introduction of either a CEN TRM140 or a CEN TRM1 plasmid. Since the growth defect is observed in the presence of cycloheximide, which is known to affect the translocation step of translation (McKeehan and Hardesty 1969; Schneider-Poetsch et al. 2010) and requires a strain lacking both m3C32 and m2,2G26 (Ellis et al. 1986), it is highly likely that growth is affected due to impaired function of one or more tRNA species during translation. Since these two modifications occur in different regions of the tRNA, one simple interpretation of this result is that the modifications each partially impair function of one or more particular tRNAs in different ways and that the combined result is poor translation by those tRNAs. Alternatively, it is possible that both modifications alter the same property of the target tRNA by each impairing translocation or another step in translation, much like the Hirsh suppressor mutation in the D-stem affects translation fidelity in the anti-codon (Hirsh 1971).

It is tempting to speculate that the programed +1 frameshift has a biological function or is part of a regulatory loop, much as the +1 frameshift required for synthesis of the S. cerevisiae Oaz1 is triggered by high concentrations of polyamines in the cell, which then leads to inhibition of ornithine decarboxylase and lower polyamine levels (Palanimurugan et al. 2004), or as the bacterial release factor 2 (RF2) terminates its own synthesis at high concentrations of RF2 but permits +1 frameshifting otherwise (Craigen and Caskey 1986). Although the persistence of the +1 frameshift in TRM140 for 150 Myr suggests that the frameshift might constitute an important regulatory loop, the fact that the +1 frameshift appears to have been lost within the budding yeast clade by Saccharomyces castellii suggests that if the putative regulatory loop is important in budding yeast species, it is not crucial under evolutionary conditions (Farabaugh et al. 2006b). We note that the region just upstream of the frameshift in TRM140 has an unusual number of threonine codons and serine codons that require decoding by tRNAs with m3C, but we do not yet know their significance.

MATERIALS AND METHODS

Yeast strains

Strains and oligomers used to construct strains are listed in Supplemental Tables S2 and S3. Strains used for genetic tests and/or analysis of tRNA were derivatives of strain BY4741 (Open Biosystems), and strains used for analysis of biochemical activity were derived from BCY123 (Macbeth et al. 2005), obtained from M.R. Macbeth. The TRM140 (ABP140) gene was deleted in strains BY4741 or BCY123 by PCR amplification of the bleR cassette of pUG66 (Gueldener et al. 2002), using primers containing sequences directly 5′ and 3′ of TRM140 (ABP140 − 39 + Phleo and ABP140 + 38 + Phleo), followed by re-amplification with primers ABP140_UP75+Phleo and ABP140_DN75+Phleo, transformation, and selection on YPD media containing 8 μg/mL bleocin. The trm140-Δ trm1-Δ strain ySD576 was constructed by PCR amplification of the trm1-Δ:kanMX cassette from the corresponding knockout strain (Open Biosystems), using primers TRM1-300 and TRM1 + 366, followed by transformation into ySD179, and selection on YPD containing 300 μg/mL geneticin. Similar approaches were used for construction of other deletion strains, and all deletion strains were confirmed by PCR analysis using flanking chromosomal primers.

Plasmids

Plasmids and oligomers used to construct plasmids are listed in Supplemental Tables S4 and S5. CEN plasmids were made by ligation independent cloning (LIC) of appropriate DNA fragments into vectors AVA579 (CEN URA3) or AVA581 (CEN LEU2), which are derived from conventional CEN vectors by incorporation of the LIC site of BG1861 (Alexandrov et al. 2004).

For expression in yeast and affinity purification, TRM140 was cloned by LIC into vector BG2663 (2μ URA3 PGAL-ORF-tag), which is similar to BG2483 (Malkowski et al. 2007), and results in fusion of the ORF to a complex tag containing a 3C site, followed by an HA epitope, His6, and the ZZ domain of protein A. This plasmid, pSD142 (2μ URA3 PGAL-TRM140-tag), was used to make pSD242 (2μ URA3 PGAL-TRM140-frame fixed-tag), in which the translational frame of ORF YOR140W is fused directly to ORF YOR239W, by deletion of nucleotide 829 of the ABP140 (TRM140) coding sequence. This was accomplished using primers 5′ABP140-fix frame and 3′ABP140-fix frame and the Quikchange mutagenesis kit (Stratagene).

For expression in E. coli as a His6-3C-ORF, frame fixed TRM140 from pSD242 (or ORF YOR240W) was PCR amplified and cloned by LIC into expression vector AVA421 (Quartley et al. 2009).

Growth and affinity purification of Trm140p from yeast

For purification of Trm140p from yeast, yeast ORF expression plasmids pSD142 (2μ URA3 PGAL-TRM140-tag) and pSD242 (2μ URA3 PGAL-TRM140-frame fixed-tag) were transformed into strain BCY123 (Macbeth et al. 2005). Transformants were grown as described (Quartley et al. 2009), except that cells were harvested 6 h after induction with 3× YP media containing 6% galactose, yielding 5 OD-L of cells.

Crude extracts were made by bead beating, and tagged protein was purified as described (Quartley et al. 2009) by binding to IgG Sepharose, elution of bound protein with GST-3C protease, removal of protease, and dialysis in buffer containing 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, and 50% glycerol, followed by storage at −20°C.

Growth and affinity purification of His6-Trm140p, His6-ORF YOR240w, and His6-AlkB from E. coli

E. coli expression plasmids were transformed into pLys(S)BL21(DE3) cells, and transformants were grown in 25 mL LB medium containing 200 μg/mL ampicillin, at 30°C overnight, diluted 1:200, grown to an OD of 0.6 at 30°C, induced by addition of IPTG to 2 mM, and grown for 3 h at 30°C. Cells from a 1 L growth were harvested and sonicated in 8 mL of buffer containing 50 mM Tris (pH 7.5), 100 mM EDTA, 4 mM MgCl2, 5 mM DTT, 5% sucrose, 1 M NaCl, and protease inhibitors, and tagged protein was purified from crude extracts by binding to Talon resin, and elution of bound protein with 250 mM imidazole, followed by dialysis in buffer containing 20 mM Tris-Cl (pH 7.5), 200 mM NaCl, 1 mM DTT, and 50% glycerol and storage at −20°C

Isolation of bulk low-molecular-weight RNA

Yeast strains were grown in YPD or minimal media at 30°C to mid-log phase, cells were harvested in 300 OD pellets, and low-molecular-weight RNA was isolated using hot phenol, as described previously (Jackman et al. 2003). For small-scale isolation of bulk low-molecular-weight RNA, yeast cells were grown in 5 mL liquid YPD or minimal media to an OD of 2.5–3.0, and rapid RNA preps were prepared as described (Schmitt et al. 1990).

tRNA purification

tRNAThr(IGU) was purified from bulk low-molecular-weight yeast RNA using a 5′-biotinylated oligomer, 5′ Bio tRNAThr (5′ Bio-AACCGAGAUCUCCACAUUAC-3′; Integrated DNA Technologies) that is complementary to nucleotides 35–55 of tRNAThr(IGU), essentially as described (Jackman et al. 2003), followed by desalting and concentration using Amicon MLtra-4 10,000 MWCO columns (Millipore).

Primer extension assays

Oligomers used for primer extension are shown in Supplemental Table S6; 30 pmol primers were labeled with 49 pmol [γ-32P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), gel purification, phenol extraction, and ethanol precipitation. Primer extension assays were done essentially as described (Jackman et al. 2003). In a 5 μL reaction, 1–2 pmol of 5′ end-labeled primers were annealed to 2 μg of bulk RNA or 0.2 μg purified tRNA by heating to 95°C for 5 min and cooling at room temperature for 30–60 min. The annealed reaction was then extended using 0.8 U AMV reverse transcriptase (Promega) in a 10 μL reaction containing 1× AMV RT buffer and 0.11 mM of each dNTP (A, C, G, and T) for 1 h at 37°C. The extension reaction was stopped using RNA load dye (10 μL formamide containing 0.1% bromophenol blue and 0.1% xylene cyanol) and quick frozen in dry ice. The frozen reactions were heated for 5 min to 95°C, cooled on ice, and loaded on a polyacrylamide sequencing gel containing 4 M urea. The gel was dried and exposed on a phosphoimager cassette.

In vitro transcription

Plasmids EMP1568 and EMP1577 (containing tRNAThr(IGU) and tRNAPhe under control of T7 polymerase) were digested with Bst N1 for 3 h at 60°C and transcribed withT7 RNA polymerase in a 20 μL reaction mixture containing 50 mM Tris (pH 8), 1 mM spermidine, 30 mM MgCl2, 7.5 mM DTT, 4 mM ATP, 4 mM GTP, 4 mM UTP, and 0.1 mM CTP, 0.1 mg/mL DNA, and 50–100 μCi [α32P]CTP (3000 Ci/mmol from Perkin Elmer) for 1.5 h at 37°C, followed by 10% PAGE, elution of RNA, and resuspension in 10 μL TE (pH 7.5).

m3C methyltransferase assay

m3C methyltransferase activity was assayed in 20 μL reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM spermidine, 0.5 mM SAM, 5000–10,000 cpm of the corresponding specifically labeled transcript, and a source of m3C methyltransferase activity (crude extract or purified protein). Reactions were incubated for 4 h at 30°C and then stopped by the addition of 90 μL of 0.5 M Tris-HCl (pH 8.0) containing 20 μg carrier tRNA, followed by phenol extraction and ethanol precipitation of the RNA. RNA was resuspended in 5 μL solution containing 30 mM sodium acetate (pH 5.3), 0.2 mM ZnCl2, and 1 μg/μL of P1 nuclease and incubated for 1 h at 50°C, and digestion products were applied to PEI-cellulose TLC plates (EM Science) and resolved in buffer containing saturated 0.25 M lithium chloride.

HPLC analysis of nucleosides

Purified tRNAThr (1.25 μg) isolated from yeast was treated with 0.5 μg of P1 nuclease in buffer containing 30 mM sodium acetate (pH 5.2) and 0.2 mM ZnCl2 for 16 h at 37°C and then with 8 U of calf intestinal alkaline phosphatase (Roche) in 1× alkaline phosphatase reaction buffer for 1 h at 37°C. The resulting nucleosides were resolved by HPLC as described (Jackman et al. 2003).

AlkB demethylation

Purified tRNAThr (1.25 μg) isolated from yeast was treated with His6-AlkB from E. coli B (purified from a clone obtained from B. Sedgwick [Dinglay et al. 2000] using TALON Resin, as described above) in buffer containing 50 mM Hepes KOH (pH 8), 75 μM ferrous ammonium sulfate, 1 mM α-ketoglutarate, 2 mM ascorbate, and 50 μg/mL BSA for 30 min at 37°C and then stopped as described (Trewick et al. 2002).

DMS methylation reaction to generate 5′[32P]m3CMP

150,000 cpm [α32P]CTP labeled transcript was treated with DMS (1:10 dilution in 100% ethanol) in 20 μl reaction mixture containing 50 mM HEPES (pH 7.5) and 1 mM EDTA for 15 min at 70°C in a fume hood. The reaction was stopped with 300 mM sodium acetate (pH 6) and ethanol precipitated, and the pellet was resuspended and digested with 0.5 μg/μL P1 nuclease for 1 h at 50°C.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

ACKNOWLEDGMENTS

We thank Juan Alfonzo and Kirk Gaston for valuable discussions on assays, Elizabeth Grayhack and Melanie Preston for invaluable insight and discussions throughout this work, M.R. Macbeth (Carnegie Mellon University) for providing the BCY123 strain, B. Sedgwick (Cancer Research UK London Research Institute) for providing the E. coli AlkB clone, and Kimberly Dean, Josh Dewe, Michael Guy, Dan Letzring, and Joseph Whipple for comments on the manuscript. This research was supported by NIH Grant GM52347 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.2652611.

REFERENCES

  1. Aas PA, Otterlei M, Falnes PO, Vagbo CB, Skorpen F, Akbari M, Sundheim O, Bjoras M, Slupphaug G, Seeberg E, et al. 2003. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421: 859–863 [DOI] [PubMed] [Google Scholar]
  2. Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S, et al. 2004. A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics 3: 934–938 [DOI] [PubMed] [Google Scholar]
  3. Asakura T, Sasaki T, Nagano F, Satoh A, Obaishi H, Nishioka H, Imamura H, Hotta K, Tanaka K, Nakanishi H, et al. 1998. Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae. Oncogene 16: 121–130 [DOI] [PubMed] [Google Scholar]
  4. Auffinger P, Westhof E 1999. Singly and bifurcated hydrogen-bonded base-pairs in tRNA anticodon hairpins and ribozymes. J Mol Biol 292: 467–483 [DOI] [PubMed] [Google Scholar]
  5. Auffinger P, Westhof E 2001. An extended structural signature for the tRNA anticodon loop. RNA 7: 334–341 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Behm-Ansmant I, Grosjean H, Massenet S, Motorin Y, Branlant C 2004. Pseudouridylation at position 32 of mitochondrial and cytoplasmic tRNAs requires two distinct enzymes in Saccharomyces cerevisiae. J Biol Chem 279: 52998–53006 [DOI] [PubMed] [Google Scholar]
  7. Bjork GR, Jacobsson K, Nilsson K, Johansson MJ, Bystrom AS, Persson OP 2001. A primordial tRNA modification required for the evolution of life? EMBO J 20: 231–239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chernyakov I, Whipple JM, Kotelawala L, Grayhack EJ, Phizicky EM 2008. Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5′-3′ exonucleases Rat1 and Xrn1. Genes Dev 22: 1369–1380 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Craigen WJ, Caskey CT 1986. Expression of peptide chain release factor 2 requires high-efficiency frameshift. Nature 322: 273–275 [DOI] [PubMed] [Google Scholar]
  10. Dinglay S, Trewick SC, Lindahl T, Sedgwick B 2000. Defective processing of methylated single-stranded DNA by E. coli AlkB mutants. Genes Dev 14: 2097–2105 [PMC free article] [PubMed] [Google Scholar]
  11. Dunin-Horkawicz S, Czerwoniec A, Gajda MJ, Feder M, Grosjean H, Bujnicki JM 2006. MODOMICS: a database of RNA modification pathways. Nucleic Acids Res 34: D145–D149 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellis SR, Morales MJ, Li JM, Hopper AK, Martin NC 1986. Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae. J Biol Chem 261: 9703–9709 [PubMed] [Google Scholar]
  13. El Yacoubi B, Lyons B, Cruz Y, Reddy R, Nordin B, Agnelli F, Williamson JR, Schimmel P, Swairjo MA, de Crecy-Lagard V 2009. The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA. Nucleic Acids Res 37: 2894–2909 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Esberg A, Huang B, Johansson MJ, Bystrom AS 2006. Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis. Mol Cell 24: 139–148 [DOI] [PubMed] [Google Scholar]
  15. Farabaugh AH, Sonawalla SB, Fava M, Pedrelli P, Papakostas GI, Schwartz F, Mischoulon D 2006a. Differences in cognitive factors between “true drug” versus “placebo pattern” response to fluoxetine as defined by pattern analysis. Hum Psychopharmacol 21: 221–225 [DOI] [PubMed] [Google Scholar]
  16. Farabaugh PJ, Kramer E, Vallabhaneni H, Raman A 2006b. Evolution of +1 programmed frameshifting signals and frameshift-regulating tRNAs in the order Saccharomycetales. J Mol Evol 63: 545–561 [DOI] [PubMed] [Google Scholar]
  17. Gao L, Bretscher A 2008. Analysis of unregulated formin activity reveals how yeast can balance F-actin assembly between different microfilament-based organizations. Mol Biol Cell 19: 1474–1484 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gerber AP, Keller W 1999. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science 286: 1146–1149 [DOI] [PubMed] [Google Scholar]
  19. Gerber A, Grosjean H, Melcher T, Keller W 1998. Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2. EMBO J 17: 4780–4789 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH 2002. A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30: e23 doi: 10.1093/nar/30.6.e23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hall RH 1963. Isolation of 3-methyluridine and 3-methylcytidine from solubleribonucleic acid. Biochem Biophys Res Commun 12: 361–364 [DOI] [PubMed] [Google Scholar]
  22. Heyman T, Agoutin B, Fix C, Dirheimer G, Keith G 1994. Yeast serine isoacceptor tRNAs: variations of their content as a function of growth conditions and primary structure of the minor tRNA(Ser)GCU. FEBS Lett 347: 143–146 [DOI] [PubMed] [Google Scholar]
  23. Hirsh D 1971. Tryptophan transfer RNA as the UGA suppressor. J Mol Biol 58: 439–458 [DOI] [PubMed] [Google Scholar]
  24. Jackman JE, Montange RK, Malik HS, Phizicky EM 2003. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9. RNA 9: 574–585 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kadaba S, Krueger A, Trice T, Krecic AM, Hinnebusch AG, Anderson J 2004. Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae. Genes Dev 18: 1227–1240 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Katz JE, Dlakic M, Clarke S 2003. Automated identification of putative methyltransferases from genomic open reading frames. Mol Cell Proteomics 2: 525–540 [DOI] [PubMed] [Google Scholar]
  27. Lecointe F, Simos G, Sauer A, Hurt EC, Motorin Y, Grosjean H 1998. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop. J Biol Chem 273: 1316–1323 [DOI] [PubMed] [Google Scholar]
  28. Lecointe F, Namy O, Hatin I, Simos G, Rousset JP, Grosjean H 2002. Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency. J Biol Chem 277: 30445–30453 [DOI] [PubMed] [Google Scholar]
  29. Ledoux S, Olejniczak M, Uhlenbeck OC 2009. A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding. Nat Struct Mol Biol 16: 359–364 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Macbeth MR, Schubert HL, Vandemark AP, Lingam AT, Hill CP, Bass BL 2005. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309: 1534–1539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Malkowski MG, Quartley E, Friedman AE, Babulski J, Kon Y, Wolfley J, Said M, Luft JR, Phizicky EM, DeTitta GT, et al. 2007. Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci 104: 6678–6683 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McKeehan W, Hardesty B 1969. The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem Biophys Res Commun 36: 625–630 [DOI] [PubMed] [Google Scholar]
  33. Olejniczak M, Uhlenbeck OC 2006. tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition. Biochimie 88: 943–950 [DOI] [PubMed] [Google Scholar]
  34. Olejniczak M, Dale T, Fahlman RP, Uhlenbeck OC 2005. Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding. Nat Struct Mol Biol 12: 788–793 [DOI] [PubMed] [Google Scholar]
  35. Palanimurugan R, Scheel H, Hofmann K, Dohmen RJ 2004. Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme. EMBO J 23: 4857–4867 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Phizicky EM, Hopper AK 2010. tRNA biology charges to the front. Genes Dev 24: 1832–1860 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pintard L, Lecointe F, Bujnicki JM, Bonnerot C, Grosjean H, Lapeyre B 2002. Trm7p catalyses the formation of two 2′-O-methylriboses in yeast tRNA anticodon loop. EMBO J 21: 1811–1820 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pruyne D, Evangelista M, Yang C, Bi E, Zigmond S, Bretscher A, Boone C 2002. Role of formins in actin assembly: nucleation and barbed-end association. Science 297: 612–615 [DOI] [PubMed] [Google Scholar]
  39. Quartley E, Alexandrov A, Mikucki M, Buckner FS, Hol WG, DeTitta GT, Phizicky EM, Grayhack EJ 2009. Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding. J Struct Funct Genomics 10: 233–247 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, et al. 2008. Lifeact: a versatile marker to visualize F-actin. Nat Methods 5: 605–607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schmitt ME, Brown TA, Trumpower BL 1990. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18: 3091–3092 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schneider-Poetsch T, Ju J, Eyler DE, Dang Y, Bhat S, Merrick WC, Green R, Shen B, Liu JO 2010. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6: 209–217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sprinzl M, Vassilenko KS 2005. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res 33: D139–D140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trewick SC, Henshaw TF, Hausinger RP, Lindahl T, Sedgwick B 2002. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419: 174–178 [DOI] [PubMed] [Google Scholar]
  45. Waas WF, Druzina Z, Hanan M, Schimmel P 2007. Role of a tRNA base modification and its precursors in frameshifting in eukaryotes. J Biol Chem 282: 26026–26034 [DOI] [PubMed] [Google Scholar]
  46. Weissenbach J, Kiraly I, Dirheimer G 1977. Primary structure of tRNA Thr 1a and b from brewer's yeast. Biochimie 59: 381–391 [DOI] [PubMed] [Google Scholar]

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