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. 2011 Jun;17(6):1111–1119. doi: 10.1261/rna.2653411

Actin-binding protein ABP140 is a methyltransferase for 3-methylcytidine at position 32 of tRNAs in Saccharomyces cerevisiae

Akiko Noma 1, Sanghyun Yi 1, Takayuki Katoh 1, Yoshimi Takai 2, Takeo Suzuki 1, Tsutomu Suzuki 1
PMCID: PMC3096043  PMID: 21518805

Abstract

Transfer RNAs contain various modified nucleotides that are introduced enzymatically at the post-transcriptional level. In Saccharomyces cerevisiae, 3-methylcytidine (m3C) is found at position 32 of the tRNAs for Thr and Ser. We used a systematic reverse genetic approach combined with mass spectrometry (ribonucleome analysis), and identified the actin-binding protein ABP140 as the protein responsible for m3C formation in both tRNAThr1 and tRNASer1. ABP140 consists of an N-terminal actin-binding sequence and a C-terminal S-adenosylmethionine (Ado-Met) binding motif. Deletion of the actin-binding sequence in ABP140 did not affect m3C formation, indicating that subcellular localization of ABP140 to actin filaments is not involved in tRNA modification. m3C formation in tRNAThr1 could be reconstituted using recombinant Abp140p in the presence of Ado-Met, whereas m3C did not form in tRNASer1 in vitro, indicating the absence of a factor(s) required for tRNASer1 m3C formation. Thus, ABP140 has been designated TRM140 according to the preferred nomenclature. In addition, we observed a specific reduction of m3C formation in HeLa cells by siRNA-mediated knock down of the human ortholog of TRM140.

Keywords: 3-methylcytidine, ABP140, S-adenosylmethionine, TRM140, tRNA

INTRODUCTION

RNA modifications play pivotal roles in the biogenesis, metabolism, structural stability, and functions of RNA molecules. To date, more than 100 chemically different, site-specific RNA modifications have been identified (Dunin-Horkawicz et al. 2006; Cantara et al. 2011). The majority of RNA modifications occur in tRNAs (Juhling et al. 2009). In particular, a wide variety of modifications in the anticodon loop of tRNAs facilitate the efficient and accurate recognition of cognate codons, and are therefore integral in protein synthesis (Bjork 1995; Curran 1998; Suzuki 2005; Agris et al. 2007).

3-methylcytidine (m3C) (Fig. 1A) was first isolated in 1963 from soluble yeast RNA (Hall 1963). m3C is found in both eukaryotic rRNAs and tRNAs (Iwanami and Brown 1968; Maden and Salim 1974). In Saccharomyces cerevisiae, m3C is located at position 32 of tRNAThr1 and tRNASer1 (Fig. 1B; Weissenbach et al. 1977; Olson et al. 1981). In the anticodon loop of tRNAs, residues at position 32 at the 5′ end of the anticodon loop are nearly conserved as pyrimidines (Y = U/C). Y32 forms a noncanonical base pair with the residue at position 38 (Olejniczak and Uhlenbeck 2006), which is most frequently adenosine. It has been proposed that the structure of the Y32–A38 pair is essential for the U-turn conformation of the anticodon loop. A U32C mutation in Escherichia coli tRNAGly resulted in tighter binding to both cognate and near-cognate codons at the A-site of the ribosome (Lustig et al. 1993). Similarly, misincorporation of Ala at the near cognate GUC codon was caused by tRNAAla with mutations of base pairs at positions 32 and 38 (Murakami et al. 2009). These observations clearly suggest the importance of the Y32–A38 pair of aminoacyl-tRNAs for uniform and accurate codon recognition at the A-site (Olejniczak and Uhlenbeck 2006). At the P-site of the ribosome, the C-terminal tail of the ribosomal protein S9 contacts residues 32–34 of peptidyl-tRNAs (Korostelev and Noller 2007; Nasvall et al. 2009). Aberrant interaction between the C-terminal tail of S9 and residue 32 resulted in an increase in +1 frameshifting, indicating that the correct recognition of residue 32 by S9 has a critical role in reading-frame maintenance. Despite the functional importance of position 32, little is known about m3C32. Methyl modification of m3C32 might modulate accurate codon recognition at the A-site and the proper ribosomal positioning at the P-site. To investigate the functional roles of m3C, it will be necessary to identify the gene or enzyme responsible for this modification.

FIGURE 1.

FIGURE 1.

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The chemical structure of 3-methylcytidine and its position in tRNAThr1 and tRNASer1. (A) Chemical structure of 3-methylcytidine (m3C). (B) Secondary structures of S. cerevisiae tRNAThr1 and tRNASer1 with modified nucleosides: 3-methylcytidine (m3C), N2-methylguanosine (m2G), dihydrouridine (D), N2,N2-dimethylguanosine (m22G), inosine (I), N6-threonylcarbamoyladenosine (t6A), pseudouridine (Ψ), 5-methylcytidine (m5C), 5-methyluridine (m5U), 1-methyladenosine (m1A), N4-acetylcytidine (ac4C), 2′-O-methylguanosine (Gm), 5-carbamoylmethyluridine (ncm5U), N6-isopentenyladenosine (i6A), and 2′-O-methyluridine (Um). Arrows indicate sites of RNase T1 cleavage to generate RNA fragments containing residues at position 32.

To explore the genes responsible for tRNA and rRNA modifications, our group has developed a genome-wide screen using a reverse genetic approach combined with mass spectrometry, which we call “ribonucleome analysis” (Suzuki 2005; Suzuki et al. 2007). This analysis utilizes a knockout strain collection of S. cerevisiae (or E. coli). Total RNA extracted from each strain is analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine whether deletion of a particular gene leads to the absence of a specific modified nucleoside, thereby permitting us to identify the enzyme or protein responsible for the modification. In the case of essential genes, temperature-sensitive mutants were cultured at the nonpermissive temperature, or expression-controlled strains were used (Soma et al. 2003). Ribonucleome analysis allows us to identify not only the enzymes directly responsible for RNA modifications, but also genes that encode proteins that lack enzymatic activity but are necessary for RNA modifications. These include carriers of the metabolic substrates used for RNA modifications and partner proteins needed for RNA recognition. Using this approach in yeast, we identified and characterized TYW1-4 for wybutosine (Noma et al. 2006) and URM1, UBA4, NCS2, NCS6, and TUM1 for 2-thiouridine in S. cerevisiae (Noma et al. 2009).

In this study we utilized ribonucleome analysis to determine the enzyme responsible for m3C formation in tRNAThr1 and tRNASer1, and identified ABP140, which encodes an actin-binding protein with previously unknown function (Asakura et al. 1998).

RESULTS

Genome-wide screening for the gene responsible for m3C formation by ribonucleome analysis

To explore the genes responsible for tRNA modifications, including m3C in S. cerevisiae, ribonucleome analysis was performed (Suzuki et al. 2007). Assuming that the gene responsible for m3C formation was nonessential, 4829 nonessential genes in S. cerevisiae were examined as a parent population for screening. Because m3C is also found in Schizosaccharomyces pombe, 3482 genes were selected for study, the orthologs of which are present in S. pombe. Finally, 351 genes whose ORFs were described as proteins of unknown function in S. cerevisiae (CYGD: http://mips.gsf.de/genre/proj/yeast) (Guldener et al. 2005) were chosen for ribonucleome analysis. In the mass chromatogram (Fig. 2A), m3C was detected as a proton adduct form (MH+; m/z 258) in wild-type cells. Among the 351 haploid knockout strains, the yor240wΔ was identified as a strain in which m3C was absent (Fig. 2A). YOR240w encodes the latter half of a long gene named ABP140 (Fig. 3A). ABP140 was reported as a fused gene of YOR239w and YOR240w by connecting with the +1 frameshift site at the junction of these two genes (Asakura et al. 1998). We next examined the yor239wΔ strain, which lacks the former half of this gene (Fig. 3A) and found the absence of m3C (Fig. 2A). We also constructed an abp140Δ strain that lacks the entire ABP140 gene. As expected, m3C was absent in the abp140Δ strain (Fig. 2A). Levels of other detectable nucleosides were normal in these strains (data not shown).

FIGURE 2.

FIGURE 2.

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Mass spectrometric analysis of total nucleosides and individual tRNAs from S. cerevisiae wild-type and mutant cells. (A) LC/MS analysis of total nucleosides from strains of wild-type (WT), yor239wΔ, yor240wΔ, and abp140Δ. Graphs show mass chromatograms traced by MH+ (m/z 258) of m3C, m5C, and 2′-O-methylcytidine (Cm). Arrows indicate the retention time for m3C. (B,C) CapLC/nanoESI MS analysis of RNase T1-digested tRNAThr1 (B) or tRNASer1 (C) isolated from wild-type (top) and yor240wΔ (bottom) strains. (Left) The mass chromatograms traced by doubly charged ions of fragments bearing m3C32 (m/z 1602 [B] or 1679 [C]) and C32 (m/z 1595 [B] or 1665 [C]). (Right) The mass spectra of position 32 containing fragments. The charge states of multiply charged ions are indicated in parentheses. The RNA sequences of each fragment are indicated on each graph. “>p” at the 3′ end of the fragment indicates cyclic phosphate. “Relative abundance” for y-axis of mass chromatograms and mass spectra shown in this figure and Figures 4, 5, and 6 stands for the percentile presentation of peak intensities that were normalized, with the highest peak defined as 100.

FIGURE 3.

FIGURE 3.

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Domain structure and sequence alignment of the ABP140/TRM140 protein and its homologs. (A) Domain structure of the S. cerevisiae ABP140/TRM140 protein. Positions of YOR239w and YOR240w are shown. The actin-binding motif, +1 frameshift site, and Ado-Met binding motif are indicated. (B) Sequence alignment of ABP140 and its homologs from fungi and other eukaryotes. All of these homologs were previously indicated (Farabaugh et al. 2006). The conserved actin-binding site and Ado-Met binding motif are underlined. The 7-mer slippage sequence is indicated at the +1 frameshift site. Motifs I–VI of Rossmann-fold-type class I methyltransferases are lined at the top of the sequence. The residues and C-terminal conserved region mutated in this study are indicated. Accession numbers are NC_006033.1 (Candida glabrata), NP_983532 (Ashbya gossypii), CAB76043.1 (Schizosaccharomyces pombe), BAE31989 (Mus musculus), NP_060866 (Homo sapiens), and NP_647636 (Drosophila melanogaster).

Absence of m3C at position 32 in tRNAThr1 and tRNASer1

To confirm the absence of m3C at position 32 of individual tRNAs, tRNAThr1 and tRNASer1 were isolated from both wild-type and yor240wΔ strains. The absence of m3C in both tRNAs isolated from yor240wΔ strain was confirmed by nucleoside analysis using LC/MS (Supplemental Fig. 1). Next, each tRNA was digested into RNA fragments by RNase T1 and analyzed by capillary liquid chromatography nano electrospray mass spectrometry (CapLC/nanoESI MS) (Suzuki et al. 2007; Ikeuchi et al. 2010). In wild-type tRNAThr1, the RNA fragment containing position 32, Cm22GCCACAm3CUI>p (MW 3206.4), was detected as doubly, triply, and quadruply charged ions (m/z 1602.1, m/z 1067.9, and m/z 800.8) as expected (Fig. 2B). The equivalent RNA fragment in tRNAThr1 isolated from the yor240wΔ strain was detected as m/z 1595.1 (a doubly charged ion) with a MW of 3192.4, which exactly matched with the molecular mass of the RNA fragment with C32 (Cm22GCCACACUI>p). In wild-type tRNASer1, the wobble base has not yet been reported (Olson et al. 1981). Therefore, nucleoside analysis was performed, which resulted in the identification of the wobble base as 5-carbamoylmethyluridine (ncm5U) (Fig. 1B; Supplemental Fig. 1). The RNA fragment containing position 32, Am3CUncm5UGp (MW 1680.2), was detected as singly and doubly charged ions (m/z 1679 and m/z 839) (Fig. 2B). The equivalent RNA fragment in tRNASer1 isolated from the yor240wΔ strain was detected as m/z 1665 (MW 1666.2), which corresponded to the molecular mass of the RNA fragment with C32 (ACUncm5UGp). To confirm this result, primer extension analysis was performed to verify the absence of m3C in tRNASer1 from the yor240wΔ strain (Supplemental Fig. 2). In addition, both tRNAThr1 and tRNASer1 isolated from the yor240wΔ strain lacked only m3C at position 32 and retained other modifications (data not shown; pseudouridines were not analyzed).

The Ado-Met binding motif in ABP140 is required for m3C formation

S. cerevisiae ABP140 consists of an N-terminal actin-binding motif, a +1 frameshift site, and the C-terminal Ado-Met binding motif (Fig. 3A). When aligned with orthologs from other organisms (Fig. 3B), the C-terminal Ado-Met binding motif is conserved in budding yeast, fission yeast, Drosophila, and mammals, while the N-terminal region is only conserved in budding yeast, including Candida glabrata and Ashbya gossypii (Fig. 3B).

At the +1 frameshift site (Fig. 3A), the sequence CTTAGGC is a slippery sequence that can promote ∼40% frameshift efficiency (Belcourt and Farabaugh 1990; Shah et al. 2002; Farabaugh et al. 2006). On the 0-frame, CTT-AGG is translated as Leu-Arg. Upon +1 frameshift, CTTA-GGC is recoded to Leu-Gly. To test whether or not the frameshift event is required for m3C formation, the abp140Δ strain was transformed with a plasmid vector (WT(0-frame)) harboring ABP140, in which the +1 frameshift site (CTTAGGC) was replaced with the 0-frame sequence (CTTGGC). Because m3C was efficiently formed with this construct (Fig. 4), the +1 frameshift event is not associated with m3C formation.

FIGURE 4.

FIGURE 4.

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Complementation of the yor239wΔ strain with plasmid-encoded ABP140 containing various mutations. LC/MS analysis of total nucleosides isolated from yor239wΔ strains with various ABP140 constructs containing various mutations; WT(0-frame), delN, D466A, F481A, D547A, K559A, Y569A, and del(D602-Q621). Mass chromatograms detecting MH+ (m/z 258) of m3C, m5C, and Cm are shown. Arrows indicate the retention time for m3C.

Abp140p localizes to actin filaments in the cell. Next, whether or not binding of Abp140p to actin filaments is required for m3C formation was examined. An ABP140 mutant (delN) lacking the N-terminal actin-binding motif was constructed (Fig. 3B), and the cellular localization of delN was analyzed by fluorescent microscopy. The delN mutant was diffused in cytoplasm (data not shown) and, as shown in Figure 4, m3C in the abp140Δ strain was efficiently formed. Therefore, localization of Abp140p to actin filaments is not necessary for m3C formation in tRNAs.

ABP140 contains a conserved Ado-Met binding motif in its C-terminal region. To determine the role of this motif in m3C formation, five conserved amino acids were substituted with Ala (D466A, F481A, D547A, K559A, and Y569A), and the conserved region near the C terminus was deleted (delD602-Q621) (Fig. 3B). The abp140Δ strain was then transformed with each of these constructs. As shown in Figure 4, no m3C formation was observed in the D466A and delD602-Q621 mutants, and the D547A mutant showed decreased activity. Other mutants efficiently accomplished m3C formation. These data demonstrated that the conserved Ado-Met binding motif in ABP140 is involved in m3C formation.

In vitro reconstitution of m3C with recombinant Abp140p

Next, we performed in vitro reconstitution of m3C using recombinant ABP140 protein. A hexahistidine-tagged Abp140p was expressed in S. cerevisiae and purified using a Ni2+-chelating column. Although the molecular weight of Abp140p was calculated to be 71.5 kDa based on its amino acid sequence, Abp140p is detected at around 140 kDa by SDS-PAGE analysis (Asakura et al. 1998). Likewise, the recombinant Abp140p was detected at ∼150 kDa due to the 19-kDa C-terminal tag (Fig. 5A), indicating a homodimer formed through covalent linkage(s) other than disulfide bond. Purified Abp140p was confirmed by peptide mapping using LC/MS (data not shown). For in vitro reconstitution of m3C, tRNAThr1, and tRNASer1 purified from the yor240wΔ strain were utilized as substrate tRNAs. When tRNAThr1 was used as a substrate, m3C was clearly introduced by the recombinant Abp140p in the presence of Ado-Met (Fig. 5B). On the other hand, tRNASer1 was not methylated by Abp140p under the same condition as the methylation of tRNAThr1 (Fig. 5B), indicating that some essential factor(s) required for m3C formation in tRNASer1 was missing. Alternatively, the order of modifications may be important for m3C32 formation in tRNASer1, potentially indicating that the introduction of other modification(s) prior to m3C32 might inhibit m3C32 formation. We then used unmodified tRNASer1 that was transcribed in vitro using T7 RNA polymerase as a substrate for Abp140p. No m3C32 formation activity was observed in this transcript (data not shown). Some modifications in tRNASer1 might act as positive determinant for m3C32 formation. In the case of the tRNAThr1 transcript, very little m3C formation could be observed in this transcript (data not shown). This result suggested that other modifications are required for efficient m3C32 formation in tRNAThr1.

FIGURE 5.

FIGURE 5.

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In vitro reconstitution of m3C32 by recombinant Trm140p. (A) C-terminal hexahistidine-tagged recombinant Trm140p resolved on SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Mass chromatograms detecting MH+ (m/z 258) of m3C (and m5C) in total nucleosides from individual tRNAThr1 (top left) and tRNASer1 (bottom left) isolated from the wild-type strain. (Right) The same mass chromatograms (m/z 258) of tRNAThr1 (top right) or tRNASer1 (bottom right) in the absence (−Ado-Met) or presence (+Ado-Met) of Ado-Met.

These results demonstrated that Abp140p is a methyltransferase for m3C32 of tRNAThr1. Therefore, Abp140p was designated as Trm140p according to the preferred nomenclature.

METTL2B is a human ortholog of Trm140p

Mammalian homologs of Trm140p were searched by BLAST with ABP140 as a query. The search retrieved methyltransferase-like protein 2B (METTL2B) and methyltransferase-like protein 2 as human and mouse orthologs, respectively (Fig. 3B). Phylogenic analysis of these homologs were reported (Farabaugh et al. 2006). Both orthologs possess the conserved Ado-Met binding motif at the C-terminal region of ABP140 and lack the N-terminal region of ABP140. To examine whether METTL2B is a methyltransferase for m3C formation, short-hairpin(sh)RNA was used to knock down METTL2B mRNA in HeLa cells. Five days after transfection of shRNA, the cells were harvested and total RNAs were extracted. LC/MS analysis revealed that the steady-state level of m3C was specifically decreased upon knock down of METTL2B (Fig. 6). Therefore, METTL2B was detemined to be a human homolog of Trm140p. However, there might be some partner proteins of METTL2B that are required to reconstitute m3C formation in tRNAs. Further investigation still needs to clarify this issue.

FIGURE 6.

FIGURE 6.

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Knockdown of human TRM140 decreases m3C formation. LC/MS nucleoside analysis of RNA from cells treated with shRNAs targeting GFP (left) and hTRM140 (right). (Top, bottom) Mass chromatograms detecting MH+ (m/z 258) and BH2+ (m/z 126) of m3C and m5C, respectively.

DISCUSSION

ABP140 was first isolated as an actin-binding protein from the soluble fraction of S. cerevisiae (Asakura et al. 1998). Abp140p interacts with filamentous(F)-actin and has an activity to cross-link F-actin into a bundle in vitro. In the cell, Abp140p colocalizes with cortical actin patches and cytoplasmic actin filaments. However, disruption of the ABP140 gene did not affect cell growth or organization of F-actin. The functional role of ABP140 has been elusive. ABP140 fused to GFP has been used as a reporter to visualize F-actin in the cell (Yang and Pon 2002). The N-terminal 17-mer peptide of ABP140 was found to be an actin-binding motif sufficient to mediate actin localization (Riedl et al. 2008), and a chemically modified 17-mer peptide has been developed as a versatile tool by which to visualize actin dynamics in the cell (Riedl et al. 2008). Although ABP140 shows a functional relevance to F-actin, we found no significant importance for actin localization in m3C formation, as no effect on m3C formation was observed upon deletion of the N-terminal actin-binding motif. In addition, in an in vitro m3C formation in tRNAThr1 catalyzed by recombinant Trm140p, the addition of F-actin to the reaction mixture did not alter of m3C-forming activity (data not shown). Taken together, the actin-binding activity of Trm140p appears to be independent of m3C catalytic activity.

From the viewpoint of tRNA biogenesis, actin localization of Trm140p might be involved in hierarchical modification of tRNAs. Most of the modifications are introduced into tRNA precursors in the nucleus (Knapp et al. 1978; Phizicky and Hopper 2010), since many tRNA-modifying enzymes are localized there. Some modifications, however, can be introduced into tRNAs after export to the cytoplasm (Murthi et al. 2010). Considering the actin localization of Trm140p, m3C formation should occur in the cytoplasm. In fact, tRNAThr1 and tRNASer1 isolated from the trm140Δ strain contained most of the modifications other than m3C32 (data not shown, pseudouridines were not analyzed), indicating that m3C32 is introduced at the late step of tRNA biogenesis in the cytoplasm. This is consistent with our observation that the unmodified tRNAThr1 transcript was not as good a substrate for Trm140p, while the modified tRNAThr1 isolated from the trm140Δ strain was efficiently methylated by Trm140p. Hence, Trm140p might monitor tRNA maturation in the cytoplasm by recognizing other tRNA modifications.

Mass spectrometric analysis of individual tRNAs isolated from the trm140Δ strain revealed that TRM140 is involved in m3C formation in both tRNAThr1 and tRNASer1. However, tRNASer1 was not methylated by recombinant Trm140p in vitro under the same conditions that allow it to methylate tRNAThr1 (Fig. 5B). This suggests that there might be partner protein(s) for Trm140p that are required for it to recognize tRNASer1 specifically. According to interactome analysis (Stark et al. 2011), there are 22 proteins that can associate with Abp140p (Trm140p). In addition, actin-binding proteins might also be candidates for partners of Trm140p. Further study will be necessary to clarify how tRNASer1 is methylated by Trm140p. Otherwise, the order of modifications might be important for m3C32 formation in tRNASer1. Considering efficient m3C32 formation in tRNAThr1 isolated from trm140Δ strain, there might be some modifications that act as positive determinants for m3C32 formation in tRNASer1.

Because position 32 of tRNA has an important role in accurate codon recognition, m3C32 formation in tRNAThr1 or tRNASer1 might play a role in modulating codon recognition and translational efficiency. No obvious phenotype of trm140Δ has been detected (Asakura et al. 1998), which indicates that this modification has no critical effect on translation. Recently, a chemical genetic approach was applied to characterize S. cerevisiae gene-deletion strains (Alamgir et al. 2010). In this study, a trm140Δ strain showed increased sensitivity to neomycin. Neomycin is an aminoglycoside antibiotic that targets the A-site decoding center on the small subunit of the ribosome. This result implies that the lack of m3C32 might affect the A-site decoding in the presence of neomycin. In addition, it will be of value to test this strain under various stress conditions, including nutrient deprivation, response to reactive oxygen species, and chemicals, to observe other phenotypes related to ABP140.

Another intriguing feature of ABP140/TRM140 is the presence of an internal frameshift site (Asakura et al. 1998) at which the slippery sequence, CUUAGGC, can promote a 40% frameshift frequency (Belcourt and Farabaugh 1990; Shah et al. 2002; Farabaugh et al. 2006). This sequence was first identified as the +1 frameshift site of the TYB gene of the Ty1 retrotransposon (Kawakami et al. 1993; Voytas and Boeke 1993). On the 0-frame, the CUU and AGG codons are decoded by tRNALeu and tRNAArg, respectively. However, the AGG codon frequently causes translational pausing at the A-site on the ribosome due to the low availability of tRNAArg encoded by a single gene. During pausing, at the P-site, tRNALeu bearing a UAG anticodon that recognizes the CUU codon, slips toward the +1 frame to base pair with the UUA codon. Then, the AGG codon for Arg is recoded as a GGC codon for Gly at the A site. Since replacing this frameshift site with the 0-frame sequence did not affect the expression of Trm140p and m3C formation (Fig. 4), the functional importance of this recoding event is still enigmatic. If m3C32 formation in tRNAThr1 or tRNASer1 modulates its capacity for codon recognition and translational efficiency, it can be speculated that expression of Trm140p might be regulated by the efficiency of +1 frameshifing in its mRNA mediated by tRNAThr1 or tRNASer1, with altered levels of m3C modification at position 32. However, these tRNAs are not directly involved in the frameshift event. In addition, the frameshift site is only conserved in very close relatives of S. cerevisiae (Fig. 3B). Even if such regulation exists, its functional importance will be limited.

ABP140/TRM140 has a conserved Ado-Met binding motifs in its C-terminal region (Fig. 3A,B). Sequence alignment based on structural motif revealed that the Ado-Met binding motif in this protein belongs to a large family of Rossmann-fold type class I methyltransferases that consist of six conserved motifs (motifs I–VI) (Fig. 3B; Kozbial and Mushegian 2005). In motif II, a highly conserved acidic residue (Asp or Glu) corresponds to Asp466 in Trm140p. When Asp466 was replaced with Ala, no m3C formation was observed (Fig. 4). According to structural analyses, this conserved Asp is located at the C terminus of β-strand 2 in Rossmann-fold-type class I methyltransferases (Martin and McMillan 2002; Kozbial and Mushegian 2005). In the crystal structure of rat glycine N-methyltransferase (GNMT) (Takata et al. 2003), the equivalent residue, Asp85, directly interacts with the 2′ and 3′ hydroxyl groups of ribose in Ado-Met via hydrogen bonds. In E. coli, when Asp277, which corresponds to Asp466 in Trm140p, was mutated in RsmC, a methyltransferase for N2-methylguanosine at 1207 in 16S rRNA, its binding affinity for Ado-Met was lost (Sunita et al. 2007). This strongly suggests that Asp466 in Trm140p is directly involved in Ado-Met binding. Mutation of Asp547 to Ala decreased m3C formation in the cell (Fig. 4). This Asp is not a conserved residue in motif VI of class I methyltransferases and might be involved in tRNA recognition. In addition, no activity of Trm140p was observed following deletion of the conserved C terminus (D602-Q621) (Fig. 4). This indicated that this region might be important for tRNA binding or protein folding. A series of mutation studies and kinetic analyses will be necessary to further characterize this enzyme. Moreover, it will also be worth attempting to obtain the crystal structure of Trm140p complexed with tRNAThr1.

MATERIALS AND METHODS

Strains, media, and plasmids

S. cerevisiae wild-type and deletion strains used in this study were obtained from EUROSCARF: the BY4742 (Mat α; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0) series and strains yor239wΔ and yor240wΔ (BY4742, YOR239w::kanMX4, and YOR240w::kanMX4). Yeast strain overexpressing hexahistidine-tagged Abp140p (YSC3869-9515832, Y258; Mat a; pep4Δ3; his4Δ580; ura3Δ53; leu2Δ3;/pABP140) was obtained from Open Biosystems. To construct the abp140Δ strain, the ABP140 ORF was replaced with URA3 by PCR-based gene deletion (Baudin et al. 1993). The PCR product was amplified from pCgURA3 (BYP1806 obtained from NBRP) as a template using the primers, 5′-ccaatacaattgtataccaaataagctattacaaattgatttaaatcgaggtcgacggtatc-3′ and 5′-cttgttttatgatgagagaggaggtggtacttgtctcagaacttccgctctagaactagtggatc-3′. The Δpep4 strain obtained from EUROSCARF [BY4741; MATa; his3 Δ 1; leu2 Δ 0; met15 Δ 0; ura3 Δ 0; pep4(YPL154c)::kanMX4] was transformed with the PCR product. Ura+ transformants were selected to obtain the abp140Δ strain. Yeast strains were grown in rich medium (YPD: 2% peptone, 1% yeast extract, and 2% glucose) or in synthetic complete medium (SC: 0.67% yeast nitrogen base without amino acids, 0.5% casamino acid, and 2% glucose) supplemented with auxotrophic nutrients as specified. The plasmid pKT10-GAL-HA-ABP140 (Asakura et al. 1998) was used for expressing Abp140p in yeast cells.

Ribonucleome analysis

The procedure was performed as described previously (Noma et al. 2006; Suzuki et al. 2007). Briefly, yeast strains were grown in 5 mL of YPD in 24-well deep well plates at 30°C and the cells were harvested during log phase growth (OD660 = 1.5–2.0). Total RNAs were extracted from the cells of each gene-deletion strain by the phenolchloroform method. Approximately 20 μg of total RNA was digested to nucleosides and analyzed by LC/MS. Absence of a specific modified nucleoside was monitored on the mass chromatogram for each strain.

Isolation and purification of individual tRNAs from yeast

Reciprocal circulating chromatography was used to isolate individual tRNAs (Miyauchi et al. 2007). The sequences of the 3′-biotinylated DNA probes complementary to yeast tRNAThr1 and tRNASer1 were 5′-tgcttccaatcggatttgaaccgatgatct-3′ and 5′-cgacaccagcaggatttgaaccagcgcggg-3′, respectively.

Mass spectrometry

RNA fragments digested by RNase T1 were analyzed by LC/MS using ion-trap MS according to a previously described method (Kaneko et al. 2003; Noma et al. 2006), with slight modifications. Purified tRNAThr1 and tRNASer1 (2.5 μg each) were subjected to digestion by RNase T1 (Seikagaku corporation; 0.6 U) for 30 min at 37°C in a buffer of 10 mM NH4OAc (pH 5.3). The hydrolysates were separated by ODS column chromatography (Xterra MS C18 2.5 μm, 2.1 x 50 mm, Waters). The solvent system consisted of 0.4 M 1,1,1,3,3,3-hexafluoro-2-propanol (pH 7.0, adjusted with triethylamine) in H2O (Solvent A) and 50% methanol (Solvent B) with the following gradients: 5% B for 0–5 min, 5%–95% B for 5–35 min, 95% B for 35–40 min, 95%–5% B for 40–42 min, and 5% B for 42–60 min. The chromatographic effluent was directly conducted to the ESI source to ionize the separated RNA fragments, which were then analyzed on an LCQ DUO ion-trap mass spectrometer (ThermoElectron). Negative ions were scanned over an m/z range of from 620 to 2000 throughout the separation.

Expression and purification of the recombinant proteins

Yeast expressing hexahistidine-tagged Abp140p was grown at 30°C in YPD. Expression of recombinant proteins was induced by the addition of 2% galactose. After cultivation for 20 h, cells were harvested and washed with ddH2O and then resuspended in lysis buffer (20 mM HEPES-KOH at pH 7.6, 10 mM KOAc, 2 mM Mg(OAc)2, 1 mM dithiothreitol [DTT] and protease inhibitor cocktail [Roche]) and disrupted by five passes through a French press (Thermo Fischer Scientific). The cell lysate was cleared by ultracentrifugation at 100,000g for 1.5 h at 4°C. The hexahistidine-tagged protein was purified with the AKTA purifier chromatography system (GE Healthcare) using a Hi-Trap chelating HP column (GE Healthcare). Briefly, the column was washed once with ddH2O and 10 mL of 0.1 M NiSO4 was loaded on the column, followed by additional washes with ddH2O. The column was then equilibrated with wash buffer (50 mM HEPES-KOH at pH 7.6, 100 mM KCl, 10 mM MgCl2, 7 mM β-mercaptoethanol, and a protease inhibitor cocktail), and the cleared cell lysate was loaded onto the column and washed with 25–50 mL of wash buffer. The His-tagged proteins were eluted from the column with a linear-gradient of imidazole (50–450 mM) in wash buffer, and the eluted fractions were analyzed by SDS-PAGE. Fractions containing full-length Abp140p were pooled and dialyzed against a buffer containing 20 mM Tris-HCl (pH 7.6), 4 mM MgCl2, 55 mM NaCl, and 1 mM DTT.

In vitro reconstitution of m3C using recombinant Trm140p

The reaction mixture (10 μL) contained 50 mM Tris–HCl (pH 8.0), 0.5 mM DTT, 10 mM MgCl2, 1 mM spermidine, 2 μg of tRNAThr1 or tRNASer1 obtained from yor240wΔ strain, and 1.4 μM recombinant Abp140p, in the presence or absence of 0.5 mM Ado-Met. The reactions were incubated for 1 h at 30°C. After incubation, the reaction was stopped by the addition of neutralized phenol. tRNAThr1 or tRNASer1 was extracted and precipitated by ethanol and then digested by nuclease P1 and bacterial alkaline phosphatase. RNA nucleoside analysis by LC/MS was carried out as described above.

RNA interference

Short hairpin RNAs (shRNAs) targeting hTrm140 and GFP (as a control) were designed by siExplorer and prepared as described (Katoh and Suzuki 2007). The sense strands of shRNAs for hTrm140 and GFP were 5′-GGAAGCAACUGACAAUGUA-3′ (shRNA-ABP140-1) and 5′-GGCACAAGCUGGAGUACAA-3′, respectively. The shRNAs were introduced into HeLa cells using Lipofectamine RNAiMAX (Invitrogen) as described previously (Katoh et al. 2009). Five days after transfection, the cells were harvested and total RNAs were extracted using TRIzol (Invitrogen). The steady-state level of hTrm140 mRNA was then measured by real-time RT–PCR using the primers 5′-ctcagcttcggtttaaaaaaggt-3′ and 5′-gtgtccagttcctcttgtgtg-3′, which confirmed that the level of hTrm140 mRNA was decreased to 8.8% of that of control cells transfected with GFP shRNA.

Site-directed mutagenesis

Site-directed mutagenesis of ABP140 was carried out in the plasmid pKT10-GAL-HA-ABP140 (Asakura et al. 1998) with QuikChange Site-Directed Mutagenesis (Stratagene) according to the manufacturer's instructions. The introduced mutation in each construct was confirmed by DNA sequencing. The following pairs of oligonucleotides were used to create each mutation:

  • WT(0-frame), 5′-gttgatgactcttgtctaggcattgatcaacagca-3′ and 5′-tgctgttgatcaatgcctagacaagagtcatcaac-3′;

  • delN, 5′-gtaccggatccggtaccatgggcgatgctaccgttgatac-3′ and 5′-gtatcaacggtagcatcgcccatggtaccggatccggtac-3′;

  • D466A, 5′-gaggataatagccgcagcttttgctccaagagccg-3′ and 5′-cggctcttggagcaaaagctgcggctattatcctc-3′;

  • F481A, 5′-ttaaaaattctgaacaggccaaccccaagtacggcc-3′ and 5′-ggccgtacttggggttggcctgttcagaatttttaa-3′;

  • D547A, 5′-taagatcatatttcgtgcttacggtgcatatgatt-3′ and 5′-aatcatatgcaccgtaagcacgaaatatgatctta-3′;

  • K599A, 5′-taactcaagttagattcgcaaagaacagaatcctag-3′ and 5′-ctaggattctgttctttgcgaatctaacttgagtta-3′;

  • Y569A, 5′-tcctagaagagaacttcgccgttagaggtgatggta-3′ and 5′-taccatcacctctaacggcgaagttctcttctagga-3′;

  • del(D602-Q621), 5′-tggaaaataaaattggaactgctgtgtttgacgttcctca-3′ and 5′-tgaggaacgtcaaacacagcagttccaattttattttcca-3′.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

ACKNOWLEDGMENTS

We thank the members of the Suzuki laboratory, in particular Y. Sakaguchi, K. Miyauchi, and S. Kimura, for their technical support and many fruitful discussions. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan (to T.S.), by a JSPS Fellowship for Japanese Junior Scientists (to A.N.), and by a grant from the New Energy and Industrial Technology Development Organization (NEDO) (to T.S.).

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2653411.

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