Mutually exclusive substrate selection strategy by human m³C RNA transferases METTL2A and METTL6

Abstract

tRNAs harbor the most diverse posttranscriptional modifications. The 3-methylcytidine (m³C) is widely distributed at position C32 (m³C32) of eukaryotic tRNA^Thr and tRNA^Ser species. m³C32 is decorated by the single methyltransferase Trm140 in budding yeasts; however, two (Trm140 and Trm141 in fission yeasts) or three enzymes (METTL2A, METTL2B and METTL6 in mammals) are involved in its biogenesis. The rationale for the existence of multiple m³C32 methyltransferases and their substrate discrimination mechanism is hitherto unknown. Here, we revealed that both METTL2A and METTL2B are expressed in vivo. We purified human METTL2A, METTL2B, and METTL6 to high homogeneity. We successfully reconstituted m³C32 modification activity for tRNA^Thr by METT2A and for tRNA^Ser(GCU) by METTL6, assisted by seryl-tRNA synthetase (SerRS) in vitro. Compared with METTL2A, METTL2B exhibited dramatically lower activity in vitro. Both G35 and t⁶A at position 37 (t⁶A37) are necessary but insufficient prerequisites for tRNA^Thr m³C32 formation, while the anticodon loop and the long variable arm, but not t⁶A37, are key determinants for tRNA^Ser(GCU) m³C32 biogenesis, likely being recognized synergistically by METTL6 and SerRS, respectively. Finally, we proposed a mutually exclusive substrate selection model to ensure correct discrimination among multiple tRNAs by multiple m³C32 methyltransferases.

INTRODUCTION

Transfer RNA (tRNA) is the most highly and diversely modified RNA species in the cell (1). To date, among 143 currently known modified ribonucleosides, 111 modifications have been identified in tRNAs from all three domains of life (2). tRNA modifications frequently occur in loop regions, such as the D-loop, TψC-loop and anticodon loop, to maintain stability of the tRNA architecture and/or guarantee fidelity and efficiency during ribosomal translation at the decoding site, thereby regulating gene expression and protein homeostasis (3,4).

3-Methylcytidine (m³C) (Supplementary Figure S1A) modification is widely found in eukaryotic cytoplasmic tRNA^Thr, tRNA^Ser, a subset of tRNA^Arg species, and mammalian mitochondrial tRNA^Thr and tRNA^Ser(UCN) at position 32 of the anticodon loop. In addition, it is present at base 20 of mammalian elongator tRNA^Met [tRNA^Met(e)] and at base 47d (e2) of mammalian tRNA^Leu(CAG) and all tRNA^Ser species (Supplementary Figure S1B) (5). Only the methyltransferases catalyzing m³C at position 32 (m³C32) of eukaryotic cytoplasmic tRNA^Thr, tRNA^Ser and tRNA^Arg have been identified (Supplementary Figure S1B) (Supplementary Table S1) (6–8). In the budding yeast Saccharomyces cerevisiae, only a single enzyme, Trm140 (ScTrm140), introduces m³C32 in both tRNA^Thr and tRNA^Ser in two different modes (6,7,9). Interestingly, ScTrm140 is expressed in fusion with an upstream actin-binding motif by a programmed +1 frameshift. However, in the fission yeast Schizosaccharomyces pombe, two separate genes encode two m³C32 methyltransferases, Trm140 (SpTrm140) and Trm141 (SpTrm141), without the actin-binding motif (10). Accordingly, SpTrm140 is no longer a dual-specificity enzyme but modifies only tRNA^Thr, while tRNA^Ser is complementarily modified by SpTrm141 (10). In mammalian cells, mouse Mettl2 and human METTL2A and METTL2B are homologous to ScTrm140/SpTrm140 and have been shown to be responsible for m³C32 formation in tRNA^Thr species (8). Recently, it has been shown that an additional cofactor, DALRD3, must interact with human METTL2A and/or METTL2B to induce m³C32 formation in human tRNA^Arg(CCU) and tRNA^Arg(UCU) species (11), although the interaction pattern and the precise role of each component in modification are still unknown. In addition, SpTrm141-homologous mouse Mettl6 catalyzes m³C32 formation in tRNA^Ser species. Interestingly, both SpTrm141 and Mettl6 interact with seryl-tRNA synthetase (SerRS) (8,9), suggesting that interaction with SerRS is an evolutionarily inherent ability of SpTrm141/Mettl6. The m³C32 modification activity of SpTrm141 is greatly stimulated by the presence of SerRS in vitro (9). However, whether SpTrm141 or Mettl6 alone could mediate m³C32 biogenesis is not yet known, and the precise interaction mode and role of either SpTrm141/Mettl6 or SerRS in tRNA binding are not fully understood. In addition, the biological function of m³C32 is poorly understood. Considering its localization in the anticodon loop, it possibly influences precise pairing between codon and anti-codon and/or biogenesis of ANG-mediated tRNA-derived fragments, as revealed by inhibition effect of m⁵C formation at C38 (12).

Although genetic data have clearly revealed the above m³C32 methyltransferases, the reconstitution of m³C32 activity using tRNA transcripts in vitro has not been successfully realized (7). Instead, in vitro m³C32 activity was achieved using tRNAs purified from a ScTrm140 gene deletion strain, suggesting that other modifications prior to m³C32 are prerequisites (7). Indeed, genetic studies have clearly demonstrated that t⁶A at position 37 (t⁶A37, catalyzed by Sua5 and KEOPS in yeasts) (13,14) or i⁶A at position 37 (i⁶A37, catalyzed by MOD5 in yeasts) (15) in specific tRNA substrates significantly triggers m³C32 biogenesis (9,10), giving an exciting example of a tRNA modification circuit (16). Therefore, to determine the tRNA recognition pattern of ScTrm140, different tRNA mutants were expressed in vivo, and the modification status at position 32 was monitored by primer extension assays, which confirmed the importance of t⁶A37 or i⁶A37 modification and revealed the key nucleotides in tRNAs (9). However, this assay does not directly determine methylation by ScTrm140, limiting the full understanding of the contributions of other nucleotides of tRNA and of the key amino acids of the methyltransferase or cofactor in m³C biogenesis.

On the other hand, the methylation of nucleotides in mRNA, such as 6-methyladenosine (m⁶A) and 5-methylcytidine (m⁵C), plays important roles in gene expression at multiple levels by influencing RNA structure and interactions within the ribosome or by recruiting specific binding proteins that communicate with other signaling pathways in physiological or pathological processes (17–19). Indeed, in addition to its presence in tRNA, m³C is present in mRNA and is suggested to be catalyzed by ScTrm140/SpTrm141/Mettl2a/Mettl6-homologous Mettl8 (8), despite a recent work reporting a dramatically lower abundance of m³C in mRNA than in tRNA (20). However, neither the specific recognition of mRNA and the mechanism of catalysis by Mettl8 nor the potential physiological or pathological role of m³C in mRNA has yet been clearly established. Therefore, studies of the substrate selection mechanism by Mettl8-homologous tRNA m³C32 methyltransferases should help to understand the mRNA m³C modification mechanism.

To understand why mammalian cells need more than one m³C32 methyltransferase and how various homologous enzymes discriminate specific tRNA substrates, in this work, using human tRNA m³C32 methyltransferases (METTL2A, METTL2B and METTL6) as models, we studied their gene expression and cellular localization; we further purified human METTL2A, METTL2B and METTL6 and prepared tRNA transcripts with and without t⁶A37 modification. We successfully reconstituted the robust m³C32 activities of both METTL2A and METTL6 in vitro, showed that METTL2B exhibited only limited methyltransferase activity in vitro and further provided a detailed tRNA selection mechanism by both enzymes.

MATERIALS AND METHODS

Materials

Anti-FLAG (F7425), anti-Myc (M4439) and anti-GAPDH (G8795) antibodies were purchased from Sigma (St. Louis, MO, USA). Anti-His₆ (AE003) was purchased from Abclonal (Shanghai, China). [³H] SAM, [³H] Arg, [¹⁴C] Thr and [¹⁴C] Ser were obtained from Perkin Elmer Inc. (Waltham, MA, USA). Dynabeads Protein G, MitoTracker and Lipofectamine 2000 transfection reagent were obtained from Thermo Scientific (Waltham, MA, USA). Primers were synthesized in Biosune (Shanghai, China), and DNA sequencing was performed by Tsingke (Shanghai, China).

Plasmid construction, expression and protein purification

Genes encoding METTL2A (UniProt No. Q96IZ6), METTL2B (UniProt No. Q6P1Q9), METTL6 (UniProt No. Q8TCB7) and SerRS (UniProt No. P49591) were amplified from cDNA obtained by reverse transcription of total RNA from human HEK293T cells. For gene expression in HEK293T cells, METTL2A and METTL2B were inserted between the Hind III and Xho I sites of pCMV-3Tag-3A and pCMV-3Tag-4A, and METTL6 was inserted between the Hind III and Xho I sites of pcDNA3.1. For gene expression in E. coli, METTL2A and METTL2B were inserted between the Sac I and Not I sites of pRSFDuet1 with an N-terminal His₆ tag, respectively. METTL6 was inserted between the SacI and NotI sites of pRSFDuet1 with an N-terminal His₆ tag. SerRS was inserted between the Nde I and Xho I sites of pET22b with a C-terminal His₆ tag. The primers used for cloning are listed in Supplementary Table S2. The METTL2A, METTL2B, METTL6 and SerRS genes were expressed in Escherichia coli BL21 (DE3) cells and induced with 200 μM isopropyl β-d-1-thiogalactopyranoside (IPTG) when the initial cell culture reached an absorbance at 600 nm (A₆₀₀) of 0.6, and transformants were cultured overnight at 18°C. Protein purification from Escherichia. coli transformants was performed with a procedure described in a previous report (21). Protein concentration was determined using a Protein Quantification Kit (BCA Assay, Beyotime, Shanghai, China) according to the manufacturer's instructions.

tRNA gene cloning and transcription

Genes encoding human cytoplasmic (hc) tRNA^Thr(AGU, CGU, UGU), tRNA^Ser(GCU), tRNA^Arg(CCU, UCU), tRNA^Asn(GUU), tRNA^Met(e) and E. coli tRNA^Thr(UGU) (EctRNA^Thr) were incorporated into the pTrc99b plasmid. tRNA transcripts were obtained by in vitro T7 RNA polymerase transcription as described previously (22,23). The overexpression and purification of EctRNA^Thr from E. coli have been described in previous reports (24,25). tRNA gene mutagenesis was performed according to the protocol provided with the KOD-plus mutagenesis kit. The primers used for template preparation are listed in Supplementary Table S2.

Determination of amino acid accepting activities

The amino acid accepting activities of various tRNAs were determined in the following reactions. A 40 μl reaction mixture containing 50 mM Tris–HCl, pH 7.5, 20 mM KCl, 10 mM MgCl₂, 2 mM DTT, 4 mM ATP, 20 μM [¹⁴C] Thr and 2.5 μM hctRNA^Thr was incubated with 2 μM mThrRS; a 40 μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 20 mM KCl, 10 mM MgCl₂, 2 mM DTT, 4 mM ATP, 20 μM [¹⁴C] Ser and 2.5 μM hctRNA^Ser(GCU) was incubated with 2 μM SerRS; a 40 μl reaction mixture containing 50 mM Tris–HCl, pH 7.5, 80 mM KCl, 12 mM MgCl₂, 2 mM DTT, 4 mM ATP, 20 μM [³H] Arg and 2.5 μM hctRNA^Arg was incubated with 2 μM ArgRS. At time intervals ranging between 15 and 60 min, aliquots of the reaction solution were added to Whatman filter pads and subsequently processed in a similar procedure with aminoacylation assays (26).

t⁶A activity assay and t⁶A modification of tRNAs

The t⁶A modification reaction was performed at 37°C in a 40 μl reaction mixture containing 50 mM Tris–HCl (pH 8.0), 200 mM NaCl, 15 mM MgCl₂, 5 mM MnCl₂, 50 mM NaHCO₃, 5 mM DTT, 4 mM ATP, 100 μM [¹⁴C] Thr, 10 μM hctRNAs or variants and 2 μM Sua5 and KEOPS.

Modification of tRNAs or variants with t⁶A was performed as follows: a 200 μl reaction mixture containing 50 mM Tris–HCl (pH 8.0), 200 mM NaCl, 15 mM MgCl₂, 5 mM MnCl₂, 50 mM NaHCO₃, 5 mM DTT, 4 mM ATP, 1 mM Thr, 50–100 μg of tRNAs or variants and 5 μM Sua5 and KEOPS. The reaction was incubated at 37°C for 1 h, and the t⁶A-modified tRNA was purified by phenol and chloroform and precipitated by EtOH with NaAc overnight at –20°C. The t⁶A-modified tRNA concentration was determined by denaturing UREA-PAGE based on linear curves from tRNA transcript samples with known concentrations.

tRNA methylation assay

The methylation reactions were performed at 37°C in a reaction mixture containing 50 mM Tris–HCl (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 10 mM spermidine, 10 mM DTT, 20 μM [³H] SAM, 5 μM transcribed, t⁶A-modified or overexpressed tRNAs, and 1 μM METTL2A, METTL2B or METTL6. At time intervals ranging between 5 and 15 min, aliquots were removed to Whatman filter pads and processed as described above.

LC-MS/MS analysis of t⁶A and m³C modified tRNA

One microgram of hctRNA^Thr(AGU), t⁶A-hctRNA^Thr(AGU), or m³C-t⁶A-hctRNA^Thr(AGU) was completely hydrolyzed by benzonase, phosphodiesterase I, and bacterial alkaline phosphatase in a 60 μl reaction containing 20 mM NH₄Ac (pH 5.2) at 37°C for 24 h. One microliter of the solution was then applied to LC–MS/MS analysis. The nucleosides were separated by HPLC on a C18 column (Agilent Zorbax Eclipse Plus C18, 2.1 50 mm, 1.8 mm) and then detected by a triple-quadrupole mass spectrometer (Agilent 6495 QQQ) in positive ion multiple reaction-monitoring mode. Mass transitions from m/z 413.1 to 281.1 (t⁶A) and m/z 258.1 to 126.1 (m³C) were monitored and recorded.

Cell culture, transfection and co-immunoprecipitation (Co-IP)

HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂ at a confluence of 70% before transfection using Lipofectamine 2000 transfection reagent according to the manufacturer's protocol. At 24 h after transfection, cells were harvested, washed with ice-cold phosphate-buffered saline (PBS) three times, and lysed with 1 ml of ice-cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100) supplemented with a protease inhibitor cocktail for 15 min at 4°C with rotation. Co-IP was performed as described previously (27,28).

Western blotting

Protein samples were separated on a 10% separating gel by SDS-PAGE and transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane, which was then blocked with 5% milk in PBST for 1 h at room temperature. Immunoblotting was performed using anti-FLAG, anti-Myc or anti-HA antibodies overnight, followed by incubation with secondary antibodies, and detected as described previously (27,28)

Immunofluorescence

HEK293T cells were transfected with specific plasmids. After 24 h, cells were stained with MitoTracker for 30 min and then fixed in 4% paraformaldehyde containing PBS for 30 min at room temperature. Fixed cells were blocked in PBS plus 0.1% Triton X-100 buffer containing 5% BSA and incubated with the primary antibody overnight at 4°C. The cells were immunostained with Alexa Fluor 488-conjugated secondary antibody in PBS for 2 h and the nuclear counterstain DAPI for 5 min at room temperature. Fluorescence images were captured with a Leica TCS SP8 STED confocal microscope.

RESULTS

Both METTL2A and METTL2B are expressed in vivo and located in the cytoplasm

In mammalian cells, METTL2A, METTL2B and METTL6 have been shown to participate in tRNA^Thr and tRNA^Ser m³C32 modification (8). Primary sequence analysis showed that the three human enzymes, together with ScTrm140 and SpTrm141, share a conserved C-terminal S-adenosyl methionine (SAM) binding domain (CTD), while a striking difference exists in the N-terminal domain (NTD) with unassigned function (Supplementary Figure S2). Compared with ScTrm140, human METTL2A and METTL2B have a large insertion (approximately 68 aa in length) in the NTD, while ScTrm141 and human METTL6 display a truncated NTD in the N-terminus. However, all the enzymes retain a conserved ‘FFKDR’ motif with an unknown role in the NTD (Supplementary Figure S2; Supplementary Figure S3A).

In humans, METTL2A (gene ID 339175) and METTL2B (gene ID 55798) are encoded by two separate genes located on chromosomes 17 and 7, respectively. However, only one gene, Mettl2 (gene ID 52686) on chromosome 11, encodes a single Mettl2 for m³C32 modification in mice. Due to the nearly identical genomic and protein sequences of human METTL2A and METTL2B (see text below), the detailed evolutionary path between the two human genes is unclear. Considering protein sequences (both 378 residues in length), only six different sites are present among the two enzymes. Three positions harbor amino acids with similar side chain properties (Val¹², Ile²⁶⁶ and Met²⁸⁸ in METTL2A vs. Ile¹², Val²⁶⁶ and Val²⁸⁸ in METTL2B), while the other three positions have completely different residues (Arg²⁶, Pro¹²⁴ and Leu¹⁵⁵ in METTL2A vs. Ser²⁶, Cys¹²⁴ and Pro¹⁵⁵ in METTL2B) (Supplementary Figure S3A).

We initially explored whether one or two genes are expressed in human cells by using liquid chromatography-tandem mass spectrometry (LC-MS) analysis of the whole cell lysis (WCL) of HEK293T cells to capture METTL2A- or METTL2B-specific peptides. Indeed, peptides spanning the same region in both METTL2A (AGSYPEGAPAVLADKR) and METTL2B (AGSYPEGAPAILDKR) were clearly detected (Supplementary Figure S3B). These data definitely showed that both METTL2A and METTL2B genes are expressed in vivo.

Furthermore, we introduced a gene encoding a C-terminal FLAG-tagged METTL2A (METTL2A-FLAG) or METTL2B (METTL2B-FLAG) separately into HEK293T cells. Immunofluorescence (IF) assays showed that METTL2A-FLAG and METTL2B-FLAG were both distributed in the cytoplasm (Supplementary Figure S3C). No clear fluorescence signal was observed in the mitochondria (Supplementary Figure S3C).

Subsequently, we purified METTL2A from E. coli cells to high homogeneity (Supplementary Figure S4A). The calculated molecular mass of purified METTL2A together with the His₆-tag should be 45.4 kDa. Its molecular mass was determined to be 34.3 kDa by gel filtration analysis with Superdex S200 based on the elution volumes of three standard proteins, apoferritin (443 kDa), yeast alcohol dehydrogenase (150 kDa) and bovine serum albumin (BSA, 66 kDa) (Supplementary Figure S4B, C). Considering that the elution volume of METTL2A was even larger than that of BSA with the smallest molecular mass among the three standards, to more accurately determine its molecular mass, a similar analysis was also performed using Superdex S75, and the molecular mass was determined to be 48.5 kDa (Supplementary Figure S4D, E) based on four standard proteins, conalbumin (75 kDa), ovalbumin (44 kDa), ribonuclease A (RNase A, 13.7 kDa) and aprotinin (6.5 kDa). These results suggested that purified METTL2A was a monomer in solution.

The above data collectively revealed that both the METTL2A and METTL2B genes are readily expressed and that METTL2A and METTL2B are located in the cytoplasm.

t⁶A37 is essential for m³C32 biogenesis of tRNA^Thr by METTL2A

m³C32 is present in tRNA^Thr, tRNA^Ser and tRNA^Arg in human cells (5). These tRNAs, including hctRNA^Thr(AGU, CGU, UGU), hctRNA^Ser(GCU) and hctRNA^Arg(CCU, UCU), were prepared by in vitro T7 run-off transcription (Figure 1A). To validate correct tRNA folding and quality, their corresponding aminoacyl-tRNA synthetases (aaRSs), including mouse cytoplasmic threonyl-tRNA synthetase (ThrRS) (26), human cytoplasmic SerRS and arginyl-tRNA synthetase (ArgRS) (29), were purified. The amino acid accepting activities of these tRNAs were approximately 1000–1500 pmol/A₂₆₀ (Figure 1A), indicating that the tRNAs were correctly folded and of high quality.

Figure 1.

t⁶A37 is essential for the m³C32 modification of hctRNA^Thr. (A) Urea gel separation of six tRNA transcripts. Their lengths and amino acid accepting activities are indicated below. (B) Time-course curves of the m³C modification of six hctRNA transcripts by METTL2A. (C) LC–MS/MS analysis of the digested products of the hctRNA^Thr(AGU) transcript, modification products by Sua5/KEOPS and subsequently by METTL2A. (D) Time-course curves of the m³C modification of the six t⁶A37-modified hctRNAs by METTL2A. Data represent averages of two independent experiments (A) or three independent experiments (B) and the corresponding standard deviation.

Subsequently, we used METTL2A and these tRNAs to reconstitute m³C32 modification activity in vitro. None of these tRNAs were modified by METTL2A (Figure 1B) [modification of hctRNA^Thr(CGU) was negligible, if any], indicating that purified METTL2A is inactive in vitro, or is active but tRNA transcripts are not suitable substrates or needs a cofactor for catalysis. ScTrm140 recognizes tRNA^Thr substrates via a sequence element including t⁶A37 (9). To understand whether t⁶A37 is a prerequisite for m³C32 formation in humans, the S. cerevisiae t⁶A modification machinery, including Sua5 and KEOPS, was purified (22). Sua5/KEOPS was able to efficiently modify all six human cytoplasmic tRNA transcripts in vitro (see text below). The tRNA^Thr(CGU) modified by Sua5/KEOPS was collected, digested with benzonase and analyzed by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS). t⁶A37 was readily detected (Figure 1C), suggesting efficient modification of t⁶A37 by Sua5/KEOPS. Then, methylation assays clearly showed that METTL2A was able to introduce m³C32 only into the three t⁶A-hctRNA^Thr species but not into t⁶A-hctRNA^Arg(CCU), -hctRNA^Arg(UCU) or -hctRNA^Ser(GCU) (Figure 1D). However, the modification levels of the three tRNA^Thr species were different, with tRNA^Thr(CGU) having the highest efficiency. In addition, LC-MS/MS analysis confirmed that the m³C32 moiety was readily decorated in the modified hctRNA^Thr(CGU) products (Figure 1C).

Thus, we successfully reconstituted m³C32 modification activity by METTL2A and revealed that METTL2A alone could modify only tRNA^Thr but not tRNA^Arg and tRNA^Ser, which requires t⁶A37 as a prerequisite.

G35 is a determinant of METTL2A for m³C32 activity reconstitution

We further explored how METTL2A discriminates among different tRNA substrates. Due to the localization of position 32 in the anticodon loop, the various anticodon loops of three hctRNA^Thrs, two hctRNA^Args and hctRNA^Ser were checked. Each of the hctRNA^Thrs, with C34, A34 or U34, could be modified, suggesting that position 34 is not a key site for modification. Among other bases in the loop, only position 35 is divergent among these tRNAs, with G35 in all tRNA^Thrs (Figure 2A). To understand its potential importance, G35 of hctRNA^Thr(CGU) (with the highest m³C32 modification efficiency) was then mutated to A35, C35 or U35. The tRNA mutants were t⁶A-modified by Sua5/KEOPS to comparable levels, suggesting that G35 is not an identity element in t⁶A modification (Supplementary Figure S5A), which was consistent with observations with the human mitochondrial t⁶A modification enzyme OSGEPL1 (22). After the preparation of t⁶A-modified tRNA^Thr(CGU) mutants, methylation determination clearly revealed that m³C32 was no longer formed in the mutants (Figure 2B), suggesting that G35 was a determinant in m³C32 biogenesis by METTL2A.

Figure 2.

G35 is a determinant of m³C biogenesis. (A) A schema showing the sequences of anticodon loops of six hctRNAs with t⁶A and m³C32 modifications (indicated in red) and two hctRNAs [hctRNA^Asn(GUU) and hctRNA^Met(e)] with only t⁶A modifications. The m³C modification levels of t⁶A-hctRNA^Thr(CGU) (black filled circles) and t⁶A-hctRNA^Thr(CGU)-G35A (blue filled squares), -G35C (green filled triangles), and -G35U (orange filled inverted triangles) (B); of t⁶A-hctRNA^Asn(GUU) (green filled circles) and t⁶A-hctRNA^Asn(GUU)-U35G mutant (blue filled squares) (C); of t⁶A-hctRNA^Met(e) (black filled circles) and t⁶A-hctRNA^Met(e)-U35G (blue filled squares) (D); and of t⁶A-hctRNA^Arg(UCU) (black filled circles) and t⁶A-hctRNA^Arg(UCU)-U35G (blue filled squares) (E) by METTL2A. Data represent averages of three independent experiments and the corresponding standard deviation.

Subsequently, in addition to hctRNA^Arg(UCU) (with C35), we also transcribed hctRNA^Asn(GUU) (with U35) and hctRNA^Met(e) (with A35), which are used to decode codons starting with A (ANN codons) and are supposed to be modified with t⁶A (note that hctRNA^Asn(GUU) and hctRNA^Met(e) do not contain m³C32 in human cells) (30). The nucleotides at position 35 of these tRNAs were also changed to G35. No impairment (instead an increase in hctRNA^Asn(GUU) and hctRNA^Met(e)) was observed in t⁶A modification by Sua5/KEOPS with wild-type and mutant tRNAs (Supplementary Figure S5B–D). However, different effects of the presence of G35 in various tRNA species were monitored; both t⁶A-modified hctRNA^Asn(GUU)-U35G and hctRNA^Met(e)-A35G clearly gained an m³C32 modification (Figure 2C, D), while m³C32 was only negligibly (if at all) introduced into hctRNA^Arg(UCU)-C35G (Figure 2E), implying that other elements in addition to t⁶A37 and G35 also critically control m³C32 formation by METTL2A.

Therefore, the above evidence showed that G35 is a critical element in m³C32 formation in tRNA^Thr species; introducing only a single G35 into a non-m³C tRNA [hctRNA^Asn(GUU) or hctRNA^Met(e)] could confer the capacity to be modified by METTL2A.

G35 and t⁶A37 are insufficient for m³C modification

The above data from hctRNA^Arg(UCU)-C35G showed that t⁶A37 and G35 alone are insufficient to confer m³C32 modification in specific tRNAs. t⁶A37 and G35 are present in E. coli tRNA^Thr species; however, m³C32 is absent in bacterial tRNAs due to the lack of m³C32 methyltransferase. To study whether METTL2A has the ability to introduce m³C32 to bacterial tRNA species, we prepared a t⁶A-modified E. coli tRNA^Thr(UGU) (EctRNA^Thr) transcript using Sua5/KEOPS. However, we found that t⁶A-containing EctRNA^Thr was not modified by METTL2A (Figure 3A). To understand whether other modifications of EctRNA^Thr were required for efficient reconstitution, we overexpressed and purified EctRNA^Thr from the E. coli MT102 strain. However, native EctRNA^Thr was likewise not a substrate of METTL2A. Notably, the t⁶A-containing hctRNA^Thr(UGU) transcript was clearly modified by METTL2A (Figure 3A). These results suggested that the tRNA sequence is the primary element leading to EctRNA^Thr hypomodification.

Figure 3.

Anticodon stems harbor critical elements for m³C32 modification by METTL2A. (A) m³C modification levels of t⁶A-hctRNA^Thr(UGU) (black filled circles), t⁶A-EctRNA^Thr(UGU) (blue filled squares) and overexpressed (O.E.) EctRNA^Thr(UGU) (orange filled inverted triangles) by METTL2A. (B) Secondary structures of EctRNA^Thr(UGU) (left) and hctRNA^Thr(AGU) (right) showing the construction of five EctRNA^Thr mutants and one hctRNA^Thr(AGU) mutant. m³C modification levels of t⁶A-EctRNA^Thr(UGU) (black filled circles) and t⁶A-EctRNA^Thr:hc-1 (blue filled squares), :hc-3 (green filled triangles), :hc-4 (orange filled inverted triangles) and :hc-5 (purple filled diamond) (C); of t⁶A-EctRNA^Thr(UGU) (black filled circles) and t⁶A-EctRNA^Thr:hc-4(blue filled squares), -27(C-G) (green filled triangles) and -31(U-A) (orange filled inverted triangles) (D); and of t⁶A-hctRNA^Thr(AGU) (black filled circles) and t⁶A-hctRNA^Thr(AGU):Ec (blue filled squares) (E) by METTL2A. EctRNA^Thr:hc-2 could not be modified with t⁶A37 and was thus not included in the methylation assay. Data represent averages of three independent experiments (A, C) or two independent experiments (D, E) and the corresponding standard deviation.

Based on the sequence of EctRNA^Thr and hctRNA^Thr(AGU) (the two tRNAs were compared because they display the highest sequence identity), we designed five EctRNA^Thr mutants by replacing some elements with their counterparts in hctRNA^Thr(AGU), including EctRNA^Thr:hc-1 (with A73U), :hc-2 (amino acid acceptor stem swapping), :hc-3 (¹⁷AGG¹⁹ replaced by ¹⁷GGU¹⁹ in the D-loop), :hc-4 (anticodon stem swapping) [note that the two stems differ only in two base pairs, A27-U43/A31-U39 in EctRNA^Thr versus C27-G43/U31-A39 in hctRNA^Thr(AGU)], and :hc-5 (C51-G63/C62 replaced by G51-C63/U62 in the TψC-stem) (Figure 3B). However, EctRNA^Thr:hc-2 was defective in t⁶A37 modification by Sua5/KEOPS, while the other mutants were modified with t⁶A37 to comparable levels (Supplementary Figure S5E). The determination of m³C32 activity showed that METTL2A indeed gained the ability to modify only EctRNA^Thr:hc-4 (Figure 3C), highlighting the determinant role of the anticodon stem. To further verify which base pairs play a key role in EctRNA^Thr:hc-4, we obtained two EctRNA^Thr mutants based on EctRNA^Thr:hc-4 [EctRNA^Thr-27(C–G) and EctRNA^Thr-31(U–A)]. m³C32 activity determination showed that both the C27–G43 and U31–A39 base pairs are needed to confer modification on EctRNA^Thr (Supplementary Figure S5F, Figure 3D). Conversely, we replaced the anticodon stem of hctRNA^Thr(AGU) with that of EctRNA^Thr (Figure 3B), and the resultant hctRNA^Thr(AGU):Ec displayed impaired but obvious t⁶A37 modification by Sua5/KEOPS (Supplementary Figure S5G) but was hypomodified by METTL2A (Figure 3E).

The above results clearly showed that in addition to G35 and t⁶A37, sequences in the anticodon stem are critical elements in determining the m³C32 activity of METTL2A. However, we found that the anticodon stem sequences are not completely conserved among human tRNA^Thr species, suggesting that they likely work collaboratively with anticodon loops and/or other elements to determine m³C32 levels in specific tRNAs, which is likely why the three human tRNA^Thr species displayed different m³C32 modification levels in vitro (Figure 1D).

The rationale of hctRNA^Ser(GCU) not modified by METTL2A

The above data clarified the key role of G35 and t⁶A37 in determining the m³C32 modification status by METTL2A. We have shown that t⁶A-hctRNA^Ser(GCU) was not modified by METTL2A in vitro. Therefore, we changed C35 of hctRNA^Ser(GCU) to G35, and the mutant hctRNA^Ser(GCU)-C35G exhibited a similar level of t⁶A modification to wild-type tRNA (Supplementary Figure S5H). Strikingly, the data showed that METTL2A could indeed introduce m³C32 into hctRNA^Ser(GCU)-C35G (Figure 4A), confirming the critical role of G35 in determining the m³C32 modification specificity of METTL2A.

Figure 4.

C35 and the long variable arm prevent hctRNA^Ser(GCU) from being modified by METTL2A. m³C32 modification levels of t⁶A-tRNA^Ser(GCU) (black filled circles), t⁶A-tRNA^Ser(GCU)-C35G (blue filled squares), -v-Thr (orange filled triangles) and -C35G-v-Thr (green filled inverted triangles) (A) and of t⁶A-hctRNA^Thr(CGU) (black filled circles) and t⁶A-hctRNA^Thr(CGU)-v-Ser (blue filled squares) by METTL2A (C). (B) Schematic diagram showing the construction of various hctRNA^Ser(GCU) and hctRNA^Thr(CGU) variable arm mutants. Data represent averages of three independent experiments (A) or two independent experiments (C) and the corresponding standard deviation.

hctRNA^Ser(GCU) differs from hctRNA^Thr predominantly in the long variable arm. To understand whether the long variable arm was also a negative element in m³C32 activity by METTL2A, we replaced hctRNA^Ser(GCU) (⁴⁴UGUGCUCUGCACGC⁴⁸) with hctRNA^Thr(CGU) (⁴⁴AGAUC⁴⁸) (Figure 4B). Despite the lack of impairment in t⁶A modification (Supplementary Figure S5H), however, the resultant hctRNA^Ser(GCU)-v-Thr was still hypomodified by METTL2A (Figure 4A). In sharp contrast, in the C35G context, hctRNA^Ser(GCU)-C35G-v-Thr showed a clearly and robustly greater m³C32 modification level than hctRNA^Ser(GCU)-C35G (Figure 4A). In parallel, we changed the variable loop of hctRNA^Thr(CGU) to that of hctRNA^Ser(GCU) (Figure 4B). No impairment in t⁶A modification level was observed (Supplementary Figure S5I); however, m³C32 modification of the mutant hctRNA^Thr(CGU)-v-Ser was significantly decreased to only slightly higher than the basal level (Figure 4C).

Taken together, these data elucidated that the absence of G35 and the presence of the long variable arm of hctRNA^Ser(GCU) precluded its modification by METTL2A.

METTL2B exhibited little m³C32 modification activity in vitro

Both the METTL2A and METTL2B genes are well expressed in vivo and have the same cytoplasmic distribution, eliciting the question of whether they display similar and redundant m³C32 modification activity. Thus, we purified METTL2B to high homogeneity (Supplementary Figure S6A). Unexpectedly, methyltransferase activity determination showed that the activity of METTL2B was only approximately 1/10 of that of METTL2A (Supplementary Figure S6B). METTL2A contains three sites (Arg²⁶, Pro¹²⁴ and Leu¹⁵⁵) that exhibit totally different side chain properties from their counterparts in METTL2B (Ser²⁶, Cys¹²⁴ and Pro¹⁵⁵). Thus, three single-point mutants, METTL2A-R26S, -P124C and -L155P, were constructed and purified (Supplementary Figure S6A). Subsequent methylation measurement showed that the activities of both METTL2A-R26S and -L155P were reduced to approximately half that of the wild-type enzyme, and the activity of METTL2A-P124C was as low as that of METTL2B (Supplementary Figure S6B). These results suggested that several natural amino acids in METTL2B, especially Cys¹²⁴, likely determined its low m³C32 methylation activity in comparison with METTL2A.

METTL6 is located in the cytoplasm and nucleus and interacts with SerRS

A previous report has shown that mouse tRNA^Ser species are modified by Mettl6 (8). We overexpressed a gene encoding C-terminal FLAG-tagged human METTL6 (METTL6-FLAG) in HEK293T cells, and IF analysis showed that METTL6-FLAG was distributed in both the cytoplasm and nucleus. Its possible mitochondrial localization was not observed (Supplementary Figure S7A). Furthermore, previous studies have shown that yeast SerRS stimulates the activity of ScTrm140 (9). METTL6-FLAG and a C-terminal Myc-tagged SerRS (SerRS-Myc) were coexpressed in HEK293T cells. By using anti-FLAG antibodies to perform Co-IP, SerRS-Myc could be precipitated with METTL6-FLAG (Supplementary Figure S7B). To understand whether the interaction is direct or indirect by relying on the presence of RNA or DNA, we then digested the DNA or RNA of whole cell lysates by DNase I or RNase A prior to immunoprecipitation. The results suggested that the interaction between METTL6 and SerRS was disrupted by RNase treatment; however, DNase I treatment was unable to abolish the interaction (Supplementary Figure S7B), suggesting that the interaction of METTL6 with SerRS depends on RNA (likely tRNA substrates).

SerRS is essential for the m³C32 biogenesis of hctRNA^Ser(GCU) by METTL6

Subsequently, we overexpressed and purified METTL6 from E. coli (Supplementary Figure S7C). We initially incubated METTL6 with t⁶A-modified hctRNA^Ser(GCU) based on the above data showing that METTL2A requires t⁶A37 for efficient m³C32 methylation. However, no methylation product was observed (Figure 5A), indicating that purified METTL6 alone is inactive or is active but requires other cofactors for modification. Considering that yeast SerRS stimulates the activity of ScTrm140 (9) and that METTL6 interacts with SerRS (Supplementary Figure S7B), we further purified human cytoplasmic SerRS (encoded by SARS1) (Supplementary Figure S7C). The inclusion of increasing concentrations of SerRS in the activity assay reaction of METTL6 (SerRS/METTL6 ranging from 1:1 to 5:1) triggered robust m³C modification; however, further elevation of SerRS from 5:1 to 10:1 decreased the m³C modification activity of METTL6 (Figure 5A). Thus, all subsequent activity determination of METTL6 was performed with SerRS at a 5:1 ratio (SerRS/METTL6). These results clearly showed that the m³C modification activity of METTL6 for tRNA, at least for hctRNA^Ser(GCU), requires the presence of SerRS. To explore whether aminoacylation or tRNA binding capacity of SerRS contributes to m³C32 modification of hctRNA^Ser(GCU), we mutated Arg³¹⁷, which is absolutely conserved in SerRSs from three domains of life and directly interacts with γ-phosphate of AMPPNP in human SerRS-AMPPNP structure (PDB No. 4RQE) but not tRNA (Supplementary Figure S8A) (31), to Ala. SerRS-R317A exhibited an abolished aminoacylation activity (Supplementary Figure S8B); however, it stimulated m³C32 activity of METTL6 to comparable levels (Supplementary Figure S8C), indicating that tRNA binding but not aminoacylation by SerRS contributed to m³C32 modification by METTL6. Subsequently, to understand whether t⁶A37 is a determinant in METTL6-catalyzed methylation, we used hctRNA^Ser(GCU) transcript as a substrate. Again, METTL6 showed no activity in the absence of SerRS (Figure 5B). In contrast to the modification of t⁶A-hctRNA^Thr(CGU) by METTL2A, the modification of the hctRNA^Ser(GCU) transcript was as robust as that of t⁶A-modified hctRNA^Ser(GCU), with similar trends concerning the relative ratio of SerRS/METTL6 (5:1 ratio with the highest efficiency) (Figure 5B). These results highlighted that the t⁶A37 moiety was nonessential and contributed little to m³C biogenesis in hctRNA^Ser(GCU) by METTL6.

Figure 5.

m³C modification activity of METTL6 for hctRNA^Ser(GCU) requires the presence of SerRS and the long variable arm. m³C modification levels of t⁶A-hctRNA^Ser(GCU) (A) and hctRNA^Ser(GCU) transcripts (B) by METTL2A without SerRS (red filled circles) and with increasing amounts of SerRS as indicated. (C) m³C modification levels of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(GCU)-C32G (blue filled squares) by METTL6-SerRS. (D) m³C modification levels of hctRNA^Ser(GCU) (black filled circles), hctRNA^Thr(CGU) (blue filled squares) and hctRNA^Arg(UCU) (green filled triangles) transcripts by METTL6-SerRS. (E) m³C modification levels of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(GCU)-v-Thr (blue filled squares), -v-Leu (green filled triangles) and -v-Sec (orange filled inverted triangles) by METTL6-SerRS; (F) Schematic diagram of constructing various variable stem and loop replacement mutants and (G) various base pair or base substitutions based on hctRNA^Ser(GCU). m³C modification levels of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(GCU)-e11 (C-G) (blue filled squares), -e12 (U-G) (green filled triangles), -e13 (C-G) (orange filled inverted triangles), -e14 (U-G) (purple filled diamond) and -ΔUe3 (red hollow circle) (H); of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(GCU)-Ue1G (blue filled squares), -Ce2A (green filled triangles), -Ue3G (orange filled inverted triangles), -e12 (A-U) (purple filled diamond) and -e12 (G-C) (red hollow circle) (I); of hctRNA^Thr(CGU) transcript (black filled circles) and hctRNA^Thr(CGU)-v-Ser (blue filled squares) (J) by METTL6-SerRS. Data represent averages of two independent experiments except three independent experiments (C) and the corresponding standard deviation.

Both positions 32 and 47d (e2) of hctRNA^Ser(GCU) contain m³C modification. We then determined which position was m³C-modified by METTL6-SerRS. To this end, we prepared a hctRNA^Ser(GCU)-C32G mutant, which was subsequently found to be hypomodified, suggesting that METTL6-SerRS is responsible for m³C32 but not m³C47d biogenesis (Figure 5C).

Rationale of the lack of modification of tRNA^Thr by METTL6-SerRS due to the lack of the variable region of tRNA^Ser as a modification determinant

Subsequently, we found no modification of hctRNA^Thr(CGU) and hctRNA^Arg(UCU) transcripts by METTL6-SerRS (Figure 5D), suggesting that METTL6-SerRS has high specificity for only tRNA^Ser.

With a typical long variable arm, hctRNA^Ser, together with hctRNA^Leu and hctRNA^Sec, constitute all the class II tRNAs in human cells. To determine any role of the long variable arm, hctRNA^Ser-v-Thr (Figure 4A) was used as a substrate in the METTL6-SerRS modification assay (Figure 5E). The results showed that the replacement of the variable arm abolished m³C32, indicating a critical role of the long variable arm. Furthermore, the variable arm of hctRNA^Ser(GCU) was changed to that of class II hctRNA^Sec or hctRNA^Leu(UAG) (Figure 5F). Similarly, the data showed that neither hctRNA^Ser(GCU)-v-Sec nor hctRNA^Ser(GCU)-v-Leu was m³C32-modified by METTL6-SerRS (Figure 5E). The above data collectively revealed that METTL6-SerRS modified hctRNA^Ser(GCU) depending on the sequence of the long variable arm.

Subsequently, we performed a sequence comparison among the variable arms of hctRNA^Ser(GCU), hctRNA^Sec and hctRNA^Leu(UAG). The variable arm of hctRNA^Ser(GCU) most resembles that of hctRNA^Leu(UAG) (Figure 5F), sharing U44, Ue1, Ce2, G47 and C48, but with a reduced size of the variable loop and a different variable stem. Targeting the different base pairs, we initially replaced each base of hctRNA^Ser(GCU) with that of hctRNA^Leu(UAG), including e11 (C–G), e12 (U-G), e13 (C-G) and e14 (U-G). Additionally, to obtain different loop sizes, we deleted Ue3 (ΔUe3) (Figure 5G). Methylation determination using the above hctRNA^Ser(GCU) mutants showed that, in comparison to wild-type hctRNA^Ser(GCU), the modification of hctRNA^Ser(GCU)-ΔUe3 and -e12 (U–G) was completely abolished; that of hctRNA^Ser(GCU)-e11 (C-G) was dramatically reduced, while that of hctRNA^Ser(GCU)-e13 (C–G) or -e14 (U–G) was not affected at all (Figure 5H). These data suggested that the e12 base pair and loop size and/or sequence were critical for m³C32 modification. We further replaced e12 (U-A) with e12 (A–U) or e12 (G–C) and constructed hctRNA^Ser(GCU)-Ue1G, -Ce2A and -Ue3G (Figure 5G). We found that a single point mutation at position Ue1 or Ce2 decreased m³C32 modification and that mutation at Ue3 abolished methylation, while the modification of hctRNA^Ser(GCU)-e12 (A–U) or -e12 (G–C) decreased to different extents (Figure 5I). It is worth noting that the Ce2A mutant from Ce2 (namely, C47d) could still be m³C-modified, again confirming that METTL6 introduces methylation at position C32 but not Ce2 (C47d).

In addition, in contrast to hctRNA^Thr(CGU), after transplanting the variable arm of tRNA^Ser, hctRNA^Thr(CGU)-v-Ser (Figure 4A) clearly gained m³C32 modification by METTL6-SerRS, although with clearly lower efficiency than hctRNA^Ser(GCU) (Figure 5J).

Altogether, these results revealed that hctRNA^Thr was not a substrate of METTL6-SerRS due to lacking the long variable arm of hctRNA^Ser; the base pairs e11 and e12 and residues e1 and e3 are among the critical nucleotide elements for the m³C32 modification of hctRNA^Ser(GCU) by METTL6-SerRS.

Anticodon loop harbors key determinants for m³C32 modification by METTL6-SerRS

METTL6 requires SerRS for m³C32 modification (Figure 5A, B) but does not directly bind it (Supplementary Figure S7B). It is well established that the long variable arm of tRNA^Ser is bound and recognized by SerRS (32). In addition, G35 is a critical determinant of methylation by METTL2A. All this evidence prompted us to explore whether the anticodon loop of hctRNA^Ser(GCU) plays a potentially important role in tRNA recognition by METTL6. To this end, we changed each base of the anticodon loop of hctRNA^Ser(GCU), except the C32 modification site (Figure 6A), resulting in the hctRNA^Ser(GCU)-U33G, -G34A, -C35G, -U36A, -A37C, and -A38C mutants. Methylation determination using the above mutants showed that, in comparison to that of wild-type hctRNA^Ser(GCU), modification of hctRNA^Ser(GCU)-U33G, -G34A, -U36A and -A37C modification was completely abolished, and hctRNA^Ser(GCU)-C35G modification was dramatically reduced, while that of hctRNA^Ser(GCU)-A38C was unexpectedly significantly increased (Figure 6B). Among the above single-point mutants, only hctRNA^Ser(GCU)-G34A and -C35G exhibited comparable levels of t⁶A modification by Sua5/KEOPS; therefore, we prepared t⁶A-modified hctRNA^Ser(GCU)-G34A and -C35G. The results showed that t⁶A-hctRNA^Ser(GCU)-G34A was still hypomodified, while the modification efficiency for t⁶A-hctRNA^Ser(GCU)-C35G was slightly elevated compared with its transcript (Figure 6B, C), suggesting that the presence of t⁶A37 compensates for the loss of the optimal anticodon sequence.

Figure 6.

The anticodon loop critically determines m³C32 formation in hctRNA^Ser (GCU) by METTL6-SerRS. (A) Schematic diagram showing the construction of six hctRNA^Ser(GCU) mutants. m³C modification levels of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(GCU)-U33G (blue filled squares), -G34A (green filled triangles), -C35G (orange filled inverted triangles), -U36A (purple filled diamonds), -A37C (red hollow circles) and -A38C (brown forks) (B); of t⁶A-hctRNA^Ser(GCU) (black filled circles) and t⁶A-hctRNA^Ser(GCU)-G34A (green filled triangles) and -C35G (orange filled inverted triangles) (C); of hctRNA^Ser(GCU) transcript (black filled circles) and hctRNA^Ser(AGA) transcript (blue filled squares) (D) by METTL6-SerRS. Data represent averages of three independent experiments (B) or two independent experiments (C, D) and the corresponding standard deviation.

hctRNA^Ser(AGA) also harbors m³C32 modification in vivo (5); however, it has a different anticodon from that of hctRNA^Ser(GCU). Subsequent modification assays showed that, in contrast to hctRNA^Ser(GCU), hctRNA^Ser(AGA) transcript was unable to be modified by METTL6-SerRS (Figure 6D). This result, together with the stimulatory role of t⁶A37 in the hctRNA^Ser(GCU)-C35G mutant, suggested that the presence of i⁶A37 modification in hctRNA^Ser(AGA) is likely a key determinant of its m³C32 modification.

In summary, in addition to the long variable arm, the anticodon loop of hctRNA^Ser(GCU) contains key elements that determine m³C32 biogenesis by METTL6-SerRS.

The NTD and CTD domains of METTL2A and METTL6 are mutually incompatible

METTL2A and METTL6 share highly similar CTD domains. However, the NTD domain of METTL6 is sharply truncated or degenerated compared with that of METTL2A. Which domain or element in enzymes determines their totally distinct substrate specificity is unclear. Therefore, we switched the corresponding NTD domains to understand whether the substrate preference could be artificially altered. We fused the NTD of METTL6 with the CTD of METTL2A to obtain N6-METTL2A; similarly, N2-METTL6 was also constructed (Supplementary Figure S9A).

We purified both N6-METTL2A and N2-METTL6 from E. coli. In vitro methylation assays showed that neither enzyme was able to introduce methylation at t⁶A-hctRNA^Thr(CGU) (Supplementary Figure S9B), suggesting that both the METTL2A NTD and CTD domains are critical for its m³C32 modification activity. In the presence of SerRS, we also determined the modification of t⁶A37-hypomodified or t⁶A37-modified hctRNA^Ser(GCU) by the two chimeric enzymes. Similarly, no modification was observed (Supplementary Figure S9C, D). In addition, considering that METTL2A requires G35 as a positive determinant, we also modified t⁶A-hctRNA^Ser(GCU)-C35G; again, the two chimeric enzymes generated no m³C32 (Supplementary Figure S9E).

Furthermore, we expressed the genes encoding METTL2A, METTL6, N6-METTL2A and N2-METTL6 in HEK293T cells. However, the two chimeric mutants were not detected in the WCL, probably due to inefficient expression or rapid degradation after synthesis (Supplementary Figure S9F).

Above all, these data collectively suggested that the NTD and CTD domains of METTL2A and METTL6 were mutually incompatible in expression/stability in vivo and for m³C32 modification in vitro.

DISCUSSION

Only a single Mettl2 is present in some eukaryotes, such as mice; however, the simultaneous existence of two nearly identical m³C32 methyltransferases with the same cellular distribution, METTL2A and METTL2B, in others, such as human, is puzzling (8). We showed here that the activity of METTL2B is far lower than that of METTL2A. Notably, among the three different variations between the two enzymes, alternative rigid Pro residue is frequently observed. Indeed, the P124C mutation alone in METTL2A is sufficient to dramatically reduce its activity to levels comparable to those of METTL2B. Considering that DALRD3 is required for m³C32 formation in tRNA^Arg(CCU) and tRNA^Arg(UCU) isoacceptors in human cells (11), it is likely that these amino acid variations between two enzymes, especially the presence or absence of rigid Pro, frequently function in determining local/global protein conformation and fine-tune protein conformation and/or structure, which may influence their interaction with DARLD3 to control the tRNA^Arg methylation level. Alternatively, METTL2B possibly needs other unknown co-factors in catalyzing tRNA modification. On the other hand, tRNA modification has been shown to be highly dynamic in response to various stimuli or stresses (33,34). It is possible that the m³C32 modification level is precisely balanced based on the expression level or ratio of METTL2A and METTL2B, which is regulated in a tissue- or cell-specific manner.

The deficiency of METTL2A in modifying tRNA^Arg is intriguing, considering that tRNA^Arg and tRNA^Thr, both class I tRNAs, have similar secondary and tertiary structures, in contrast to class II tRNA^Ser. The presence of a tRNA binding motif in DALRD3 implies that METTL2A is inefficient in binding tRNA^Arg and is probably assisted by DALRD3. However, the exact functional assignment of METTL2A-DALRD3 remains unclear.

We also observed that once G35 is introduced into hctRNA^Ser(GCU), in the presence of t⁶A37, the tRNA mutant gains the ability to be modified by METTL2A, although further deletion of the long variable arm significantly elevates the modification level. However, in human cells, most hctRNA^Ser species, including hctRNA^Ser(AGA), hctRNA^Ser(UGA) and hctRNA^Ser(CGA), naturally contain a G35. Sure, in these hctRNA^Ser species with G35, i⁶A37 is present instead of t⁶A37. In addition to the long variable arm, whether C35 and t⁶A37 in hctRNA^Ser(GCU) or G35 and i⁶A37 in other hctRNA^Ser species synergistically determine substrate specificity by METTL6-SerRS but not METTL2A is unknown and needs further exploration.

One remarkable difference revealed here is that METTL6 does not require modification at residue 37 as a prerequisite, at least in modification for hctRNA^Ser(GCU). On the other hand, m³C32 formation by METTL6 is dependent on the presence of SerRS. METTL6 did not directly interact with SerRS. Increasing ratio of SerRS to METTL6 from 5:1 to 10:1 decreased methylation activity of METTL6; we suggested that the affinity between components of METTL6-tRNA^Ser-SerRS ternary complex is not strong and too much SerRS might form SerRS-tRNA^Ser binary complex and thus competes with formation of ternary complex. On the other hand, the possibility of presence of trace amount of nuclease in the SerRS sample cannot be absolutely excluded. Perplexingly, a recent report shows that purified GST-tagged METTL6 alone is able to introduce m³C32 modification into total cellular RNA (35). hctRNA^Ser(GCU) transcript was used in this work. Although the possibility that other modifications in tRNA^Ser species eliminate the requirement of SerRS cannot be absolutely excluded, we suggest that the modification of total RNA may not be derived from tRNA modification. Alternatively, modifications other than hctRNA^Ser(GCU) used here, including hctRNA^Ser(AGA), hctRNA^Ser(UGA) or hctRNA^Ser(CGA), could be performed by METTL6 alone. However, when the hctRNA^Ser(UGA) transcript was used in an in vitro methylation assay, only a basal level of modification (with CPM 100–300) was observed (35). Therefore, we hypothesize that the m³C32 activity of METTL6 for hctRNA^Ser is critically dependent on SerRS. The biological function of m³C32 modification, despite being currently unidentified, should be of high significance considering that multiple tRNA methyltransferases and cofactors have evolved in human cells. Thus, SerRS is indeed a key multifaceted regulator in protein synthesis, vascular development and other functions, such as tRNA modification (36).

m³C modification has been shown to be present in mRNA (8). The independence of the t⁶A modification of METTL6 elicits another interesting question: is METTL6 able to introduce m³C to RNA species other than tRNA? To date, t⁶A modification has been detected only in tRNAs and not found in mRNAs. Therefore, in combination with our findings that METTL2A is dependent on t⁶A modification prior to m³C biogenesis, METTL6 is more likely to form m³C in RNAs other than METTL2A. Previous work has shown that yeast Trm140 recognizes SctRNA^Thrs and SctRNA^Ser(GCU) depending on t⁶A37 and SctRNA^Ser(AGA), (CGA) and (UGA) depending on i⁶A37. In addition, XGU and t⁶A37 are sufficient for m³C32 modification of yeast tRNA^Thrs (9). Our work shows that the modification of tRNA^Thrs by human METTL2A also requires an anticodon stem and that human METTL6 recognizes hctRNA^Ser(GCU) independently of t⁶A, suggesting that humans and yeasts have different mechanisms for recognizing tRNA substrates. Indeed, cellular mRNAs frequently form anticodon stem and loop-like architectures, which are responsible for recruiting interacting protein partners, such as aminoacyl-tRNA synthetases (37). In addition, some noncoding RNAs contain tRNA-like structures (38). Although a previous report indicated no obvious change in m³C abundance in the mRNA fraction in Mettl6 knockout mice (8), the frequency of m³C modification by METTL6 may be low to be accurately captured. Indeed, a recent m³C mapping analysis revealed little m³C abundance in mRNA (20).

Mettl8 has been shown to participate mRNA m³C biogenesis (8). However, nothing is known about the mRNA substrate selection mechanism of Mettl8. Mettl8 resembles METTL2A most closely in primary sequence. Our clarification of the tRNA selection recognition pattern by METTL2A, especially the key role of the anticodon stem and loop region of tRNA, provides valuable insights into how METTL8 recognizes mRNA substrates. Considering that t⁶A modification has not been detected in mRNA, whether Mettl8 does not rely on t⁶A modification is an open question. Moreover, our work showed that neither METTL6 nor METTL2A was significantly localized in mitochondria, while a recent study showed that METTL8 was localized in mitochondria (39). Therefore, we reasonably infer that METTL8 is highly likely to be responsible for the m³C32 modification of mitochondrial tRNAs.

The CTDs for binding SAM are highly conserved, while the most striking sequence difference is observed in the NTDs between METTL2A and METTL6. The NTD of METTL6 is significantly truncated only with a conserved ‘FFKDR’ motif; however, that of METTL2A is a much longer domain even than those of ScTrm140 and SpTrm140 (Supplementary Figure S2). In combination with our revealed tRNA sequence requirement for both hctRNA^Thrs and hctRNA^Ser(GCU), we proposed a model for multiple substrate selection and modification by multiple m³C32 modification enzymes (Figure 7). The CTD of both enzymes recognizes the anticodon loop region, relying on distinct sets of determinants in hctRNA^Thrs (G35 and t⁶A37) and hctRNA^Ser(GCU) (positions 33–37); the long NTD of METTL2A recognizes other elements, such as the anticodon stem, of hctRNA^Thrs. However, the degenerated NTD of METTL6 is unable to bind hctRNA^Ser(GCU) efficiently, which is augmented by SerRS for recognizing the long variable arm (including Ue3 and e12 base pairs), but they do not interact directly. METTL2A fails to modify hctRNA^Ser(GCU) due to the presence of C35 in the anticodon and the long variable arm, possibly leading to spatial conflict and/or electrostatic repulsion between the variable arm and the long NTD of METTL2A. On the other hand, hctRNA^Thrs cannot be decorated by METTL6 due to an unfavorable anticodon, the lack of the long variable arm of hctRNA^Thrs (leading to inability to be captured by METTL6-SerRS) and the truncated NTD of METTL6 (making the efficient recognition of hctRNA^Thrs by METTL6 alone impossible). Notably, hctRNASer(AGA) transcripts cannot be modified by METTL6-SerRS, suggesting a distinct recognition pattern between t⁶A37- and i⁶A37-harboring hctRNA^Ser species, which should be further explored.

Figure 7.

Model of mutually exclusive substrate selection strategy by human m³C RNA transferases METTL2A and METTL6. When hctRNA^Thrs are modified by METTL2A, the key elements in the anticodon loop (t⁶A37 and G35) are recognized by the CTD, and other key elements, including the anticodon stem, are recognized by the long NTD (lower left quadrant). hctRNA^Ser (GCU) is modified by METTL6 with the assistance of SerRS, which recognizes the essential elements on the anticodon loop (bases 33–37) and variable arm (including base pair e12 and Ue3). The truncated NTD of METTL6 is unable to bind tRNA as efficiently as that of METTL2A (upper right quadrant). hctRNA^Thrs are not modified by METTL6-SerRS due to the unfavorable anticodon loop and lack of the long variable arm (upper left quadrant); in parallel, hctRNA^Ser(GCU) is not modified by METTL2A due to the lack of G35 and presence of a long variable arm (lower right quadrant). The variable arms are indicated in pink.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

Natural Science Foundation of China [31670801, 31822015, 81870896]; Shanghai Key Laboratory of Embryo Original Diseases [Shelab201904]; Key Laboratory of Reproductive Genetics, Ministry of Education, Zhejiang University [ZDFY2020-RG-0003]. Funding for open access charge: Natural Science Foundation of China [31670801, 31822015, 81870896].

Conflict of interest statement. None declared.