Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

Djemel Hamdane; Amandine Guelorget; Vincent Guérineau; Béatrice Golinelli-Pimpaneau

doi:10.1093/nar/gku820

. 2014 Sep 12;42(18):11697–11706. doi: 10.1093/nar/gku820

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

Djemel Hamdane ^1,^2,^*, Amandine Guelorget ¹, Vincent Guérineau ³, Béatrice Golinelli-Pimpaneau ^1,^2,^*

PMCID: PMC4191401 PMID: 25217588

Abstract

In most organisms, the widely conserved 1-methyl-adenosine58 (m¹A₅₈) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m¹A₅₇ being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (_PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. _PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A₅₇ but not A₅₈, confirming that _PabTrmI methylates efficiently the first adenine of the A₅₇A₅₈A₅₉ sequence. _PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m¹A₅₈ formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.

INTRODUCTION

Modified nucleosides are abundant and of wide chemical diversity in transfer RNAs (tRNAs). They influence translation accuracy, reading frame maintenance, recognition by aminoacyl-tRNA synthetases and tRNA structure (1–3). The modifications found in RNAs occur post-transcriptionally and range from simple base or ribose methylations to more complex multi-step reactions (4). Most RNA modifying enzymes are mono-site specific, introducing a chemical group at a specific position in a target nucleotide. Nonetheless, there are a few RNA modifying enzymes that display a regional multi-site specificity, modifying consecutive positions in RNA. For instance, Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase Trm1 catalyzes the methylation not only of guanine 26 but also guanine 27 in tRNA (5,6). In Escherichia coli, the pseudouridine synthase TruA specifically modifies uridines at positions 38, 39 and/or 40 in the anticodon stem loop of tRNAs (7), while rRNA MTase KsgA catalyzes the dimethylation at the N6 position of two adjacent adenines A1518 and A1519 in the small subunit of ribosomal RNA (8,9). Although the multisite specificities of these enzymes have been pointed out, no detailed mechanistic studies have yet been reported. Most of these enzymes have their structures solved but not their complexes with RNA or the RNA was in an unproductive conformation (10). When the structures in complex with RNA are known (7), they reveal the molecular features of the stabilization of the flipped base, but are less informative about the binding, base recognition and flipping processes.

The bi-specific tRNA adenine-N1, A₅₇-A₅₈-methyltransferase (MTase), named TrmI, from the archaeum Pyrococcus abyssi (_PabTrmI) methylates two adjacent adenines in tRNA (11). _PabTrmI catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to nitrogen 1 of A₅₇ and the adjacent A₅₈ in the T-loop of tRNAs. In contrast to m¹A₅₈, a conserved modification found in all organisms and shown to be essential for cell growth in yeast (12) and for adaptation to high temperatures in thermophilic organisms (13), m¹A₅₇ is exclusively encountered in archaea as a precursor of 1-methylinosine (m¹I) at position 57 (14,15). In particular, the presence of m¹A together with that of m¹I was detected after incubation of a tRNA^Ile transcript with Haloferax volcanii extracts (14), despite the fact that m¹A₅₈ has never been found in any of the 51 tRNAs of this species sequenced so far, and these two modified nucleotides were not detected with a transcript in which A₅₇ was mutated to G₅₇. Therefore, it was attested that, in vivo, the formation of m¹A₅₇ catalyzed by a SAM-dependent MTase is followed by the deamination of the 6-amino group of the adenine moiety by a currently unknown deaminase to produce m¹I₅₇.

In general, the multi-site specificity mechanism of these enzymes is not yet well understood. In the case of _PabTrmI, we have proposed that the enzyme recognizes the presence of three consecutive adenines (A₅₇A₅₈A₅₉) in P. abyssi tRNAs. Indeed, A₅₈ is methylated by _PabTrmI only if an adenine is present at position 59, suggesting that the enzyme methylates the first adenine of an AA sequence (16). Moreover, mass spectrometry (MS) analysis of methylated _PabtRNA^Asp formed by _PabTrmI indicated the presence of monomethylated A₅₇, in addition to the dimethylated product, but not that of monomethylated A₅₈, suggesting that the enzyme modifies sequentially A₅₇ and then A₅₈. This implies the existence of an RNA binding pocket with a large specificity for the nucleotide to be modified (to accommodate A or m¹A) and a rather strict one for the following nucleotide (adenine). Crystal structures of TrmIs from several organisms have been determined (17,18) but the structures in complex with RNA are still missing. Yet, the structure of TrmI from Thermus thermophilus solved in complex with S-adenosyl-L-homocysteine (SAH) revealed an active site binding pocket suited to bind a flipped-out adenine, suggesting that the enzyme uses a base flipping mechanism (19). Indeed, RNA modification enzymes commonly introduce such a structural change inside the tRNA to make the nucleoside accessible for modification (20,21). Typically, the enzyme flips the nucleobase to be modified, removing its internal stacking and hydrogen bonding interactions, to expose it to the protein active site.

The most commonly employed method for studying these local nucleotide conformational changes in solution is to replace the target base by 2-aminopurine (2-AP), a fluorescent nucleotide analog (Supplementary Figure S1). The ease, sensitivity and specificity of 2-AP fluorescence detection make the use of 2-AP very attractive. 2-AP is commonly used to probe nucleic acid structure in loops because it rarely affects structure adversely, and its fluorescence is typically enhanced when its environment is disturbed as a result of decreased stacking interactions (22). Therefore, 2-AP is an excellent probe to follow the conformational changes of the target base itself or its neighbors. Indeed, when 2-AP is stacked between DNA bases, its fluorescence is strongly quenched, but the fluorescence increases and shifts when 2-AP adopts a flipped-out position (23,24). This fluorescent analog has also been substituted to nucleobases in strategic positions of single-stranded RNA molecules to act as a probe to monitor folding and folding dynamics of RNAs in a few cases (25–28).

We applied this spectroscopic method relying on the 2-AP fluorescence to study the RNA conformational changes occurring during modification by _PabTrmI. A57 and A58 are located in the T-loop of tRNA. A58 is involved in a reverse Hoogsteen base pair interaction with U54 within the loop and is more buried inside the tRNA than A57, which is not base-paired. The difference between A and 2-AP is the removal of the amino group at position 6 and addition of an amino group at position 2 (Supplementary Figure S1). Thus, 2-AP retains the same base-pairing capacities as adenine but with a different geometry. By replacing A₅₇ or A₅₈ with 2-AP and by following the fluorescence changes during the methylation reaction, we gained deeper insight into the mechanism of _PabTrmI. In addition, we built a structural model of _PabTrmI in complex with a tRNA, which enabled us to provide a model of the sequence of events along the dual base modification.

MATERIALS AND METHODS

Material

The 2-AP containing RNAs were ordered from Thermo Fisher Scientific Dharmacon. Prior to reaction, they were refolded by heating at 80°C for 5 min, and then cooling on ice for 2 min.

Purification of _PabTrmI

The recombinant P. abyssi PAB0283 protein (_PabTrmI) bearing a C-terminal His-tag was expressed as described previously (11). After lysis, the cells supernatant was loaded onto a Nickel-NTA affinity column (5 ml; Qiagen) pre-equilibrated in 50 mM Tris-HCl pH 8.5, 500 mM KCl and eluted with the same buffer containing 200 mM imidazole. The sample was then loaded onto a Superdex 200 HR 10/30 column (GE healthcare Inc.) equilibrated in Tris-HCl, 500 mM KCl, 200 mM imidazole. The fractions containing the protein were pooled and concentrated with a Vivaspin concentrator (10 kDa PES membrane; Sartorius).

Mass spectrometry analysis

The mini-RNA (20 μM) was incubated for 1 h at 60°C with 10 μM of purified wild-type _PabTrmI and 1 mM SAM in 50 μl 50 mM Tris-HCl pH 8, 10 mM MgCl₂. The reaction was stopped with 200 μl of phenol:chloroform:isoamylic alcohol (25:24:1) pH 4.5 to precipitate the protein. tRNA in the aqueous phase was extracted by centrifugation at 10 000 g for 5 min, ethanol precipitated and desalted on a MicroSpin G-25 column (GE Healthcare). One microgram of desalted tRNA was then digested at 37°C overnight in 10 μl of 50 mM DHB (2,5-dihydroxybenzoic acid; Sigma-Aldrich, Saint Quentin Fallavier, France) containing 2 μg of RNase A (Fermentas). One microliter of digest was mixed with 9 μl DHB (20 mg/ml in water: methanol 50:50) and 1 μl of the mixture was spotted on the MALDI plate and air-dried (‘dried droplet’ method). MALDI-TOF MS and MALDI-TOF/TOF MS/MS analyses were performed directly on the digestion products using a 4800 MALDI TOF/TOF Analyzer mass spectrometer (ABSciex, Les Ulis, France). The instrument is equipped with an Nd:YAG laser (operating at 355 nm wavelength of <500 ps pulse and 200 Hz repetition rate). Acquisitions were performed in positive ion mode except those for determining the methylation kinetics that were measured in negative ion mode to ensure that the positive charge generated on the N1 atom of adenine upon m¹A formation does not affect ionization and that the percentage of methylated RNA can be accurately quantified by measuring the area of the peaks corresponding to the non-methylated and methylated fragments. For MS/MS experiments, precursor ions were accelerated at 8 kV and the MS/MS spectra were acquired using 1 kV collision energy with CID gas (argon) at a pressure of 3.5 × 10^-6 Torr. MS data were processed using DataExplorer 4.4 (Applied Biosystems).

Steady-state fluorescence assay

The emission fluorescence spectra of 2-AP containing mini-RNAs alone and in complex with _PabTrmI were recorded in a quartz cuvette at 37°C on a Cary eclipse fluorescence spectrophotometer (Varian). The excitation and emission slits were set at 5 and 10 nm, respectively. After exciting at 320 nm, the fluorescence emission was recorded every nm from 335 to 550 nm. The resulting spectra were corrected from the contribution of the buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂). In all experiments, the concentration of RNA was 0.5 μM while that of _PabTrmI was varied. Before recording the spectra, enzyme and RNA were incubated for 5 min to reach the equilibrium.

Stopped-flow kinetics

The experiments were performed on a TGK Scientific SF-61DX2 stopped-flow fluorescence spectrophotometer equipped with a temperature-controlled circulating water bath. RNA (1 μM) was rapidly mixed with _PabTrmI (6 μM) alone or in complex with SAM (1 mM) or SAH (1 mM). All reactions were performed in reaction buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂) at various temperatures. Before rapid mixing, the samples loaded in the stopped flow syringes were incubated for 10 min at the appropriate temperature. 2-AP fluorescence was recorded by exciting at 320 nm and collecting through a WG-360 nm cut-off filter. Up to five transients were collected and averaged for each condition. Background signal was determined against buffer to allow data scaling. The observed rates (k_i) were determined by fitting the averaged transients to multiple exponentials equation using the SigmaPlot 12 software. The temperature dependence of each k_i was measured and the energies of activation (E_a) were determined using the following equation: k_i = A*exp(−E_ai/RT).

RESULTS

_PabTrmI recognizes and methylates both A57 and A58 in mini-tRNA^Asp containing the A₅₇A₅₈A₅₉U₆₀ sequence

We have previously shown that _PabTrmI recognizes and methylates the first adenine of an AA sequence in a full-length tRNA (16). While _PabTrmI methylates both A₅₇ and A₅₈ in tRNA^Asp, it becomes mono-site specific with the P. abyssi A₅₉G tRNA^Asp mutant, A₅₇ being the only targeted base. To verify if the tertiary tRNA L-shaped structure is required for the multi-site specificity, we tested whether _PabTrmI methylates a 29-mer P. abyssi mini-tRNA^Asp, which includes the acceptor stem and the T-arm of P. abyssi tRNA^Asp (Figure 1A). Indeed, several RNA-modification enzymes recognize a stem-loop, in which they can efficiently extrude and modify the targeted base. We used MS to examine if the methylation has occurred and to identify the modified positions. After incubation with the enzyme and SAM, the mini-RNA was digested by RNase A, which cleaves after C and U and generates 3′-phosphate nucleosides, and its fragments were analyzed by MALDI MS (Supplementary Figure S2) (29). When incubated with _PabTrmI, the spectrum of digested mini-tRNA^Asp showed fragments at m/z 1326.15 and 1340.17 (Supplementary Figure S2A) coinciding with the expected masses of mono and dimethylated fragments derived from AAAUp, corresponding to the sequence from A₅₇ to U₆₀ in mini-tRNA^Asp (Figure 1A). The positions of the modifications at the nucleotide level were then identified by tandem mass spectrometry (MS/MS) analysis (Supplementary Figure S2B). In this technique, individual oligonucleotides of interest, which have been previously identified by standard MALDI-MS analysis, are isolated and subjected to controlled fragmentation (29). The sequence of the dimethylated fragment obtained after incubation with _PabTrmI (Supplementary Figure S2B) was determined to be m¹Am¹AAUp, indicating that both adenines at positions 57 and 58 in mini-tRNA^Asp are methylated. Indeed, the presence of the z2 fragment, corresponding to the AUp sequence (m/z 636.10), combined with the absence of the z2 + m fragment, corresponding to the m¹AUp sequence, shows that A₅₉ is not methylated. Moreover, the presence of the c2 fragment corresponding to the m¹Am¹Ap sequence (m/z 687.14) confirms that both A₅₇ and A₅₈ are methylated.

Figure 1. — Sequence and secondary structure of the mini-RNAs tested for the methylation and binding studies of _PabTrmI. (A) mini-tRNA^Asp. The fragments obtained after RNase A treatment are indicated by a dash. (B) 2AP57: mini-tRNA^Asp containing 2-AP at position 57 instead of A₅₇. (C) 2AP58: mini tRNA^Asp containing 2-AP at position 58 instead of A₅₈. (D) mini-tRNA^Arg.

_PabTrmI recognizes and methylates only A58 in mini-tRNA^Arg (TCT) containing the G₅₇A₅₈A₅₉U₆₀ sequence

Interestingly, all 43 tRNAs from P. abyssi contain either the A₅₇A₅₈A₅₉ or G₅₇A₅₈A₅₉ sequence. To confirm that _PabTrmI methylates the first adenine of an AA sequence inside the T-loop of tRNA (16), we tested the P. abyssi mini-tRNA^Arg (TCT), which possesses the G₅₇A₅₈A₅₉U₆₀ sequence (Figure 1D), as a substrate of _PabTrmI. As confirmed by MS and MS/MS analysis (Supplementary Table S1), only A₅₈ is methylated by _PabTrmI in mini-tRNA^Arg.

The 2-AP replacing the target adenines in a mini-RNA substrate is not methylated by _PabTrmI

The P. abyssi mini-tRNAs^Asp containing 2-AP either at position 57 or 58 (named as 2AP57 and 2AP58) were incubated with _PabTrmI and SAM, digested by RNase A and the resulting fragments were analyzed by MALDI MS (Supplementary Figure S3). When the adenine at position 57 was replaced by 2-AP (2AP57; Figure 1B), the mini-RNA was methylated by _PabTrmI at a unique site, as evidenced by the presence of a + 14 Da fragment (1326.07 Da compared to 1312.21 for the control experiment) (Supplementary Figure S3A). MS/MS analysis of the digested RNA identified A₅₈ as being the methylation site and indicated that the 2-AP at position 57 was not methylated by _PabTrmI (Supplementary Figure S3B). Interestingly, A₅₈ was almost completely modified, as indicated by the very small intensity of the non-methylated fragment (Supplementary Figure S3A). Similarly, when the adenine at position 58 of P. abyssi mini-tRNA^Asp was replaced by a 2-AP (2AP58; Figure 1C), _PabTrmI methylated only A₅₇ (Supplementary Figure S3C and D). However, the methylation of A₅₇ was not as efficient as that of A₅₈ in 2AP57, as demonstrated by the large amount of non-methylated fragment.

The methylation of A₅₈ is not impaired by 2-AP at the adjacent position 57

To further confirm the more efficient methylation of A₅₈ in a 2-AP-containing mini-RNA substrate, the kinetics of m¹A₅₇ and m¹A₅₈ formation were followed at 60°C with MS. While 2AP57 was methylated too fast by _PabTrmI for measuring an accurate rate constant by MS, the time course of methylation of 2AP58 by _PabTrmI yielded an observed rate constant for methylation of A₅₇ of 0.05 min⁻¹ (Figure 2). This clearly indicates a more efficient methylation of A₅₈ than A₅₇ in the 2-AP-containing mini-RNAs.

Moreover, the methylation kinetics of 2AP57 were very similar to those of the full-length P. abyssi tRNA^Asp (Figure 2).

Interaction of 2-AP-containing mini-RNAs with _PabTrmI

To know if the interaction of _PabTrmI with a 2-AP-containing mini-RNA substrate modifies the target base environment, we followed the fluorescence emission spectra from 340 to 460 nm after exciting at 320 nm 2AP57 and 2AP58 in the presence of enzyme (Figure 3). Because the fluorescence from the protein is negligible under our experimental conditions, the observed variation of fluorescence can be exclusively attributed to a change of the 2-AP environment. In the absence of enzyme, both 2AP57 and 2AP58 fluoresce with a maximum peak at ∼373 nm. Addition of a 7-fold molar excess of _PabTrmI to either 2AP57 or 2AP58 resulted in 91% and 43% fluorescence increase, respectively. This increase of fluorescence is associated with a small blue shift of the maximum peak indicating a polarity change in the 2-AP environment. The 2-AP-mini-RNA fluorescence change with increasing concentrations of _PabTrmI shows a hyperbolic shape in agreement with a binding event (Figure 3C). The apparent dissociation constants of 2AP57 and 2AP58 for _PabTrmI deduced from this experiment were 1.7 and 2.8 μM, respectively.

Figure 3. — Emission fluorescence spectra of 2AP57 (A) and 2AP58 (B) in the absence (dash lines) and presence of _PabTrmI (solid lines). The concentration of RNA was 0.5 μM and that of _PabTrmI was 6.5 μM. (C) Fluorescence titration of the 2-AP containing mini-RNAs with _PabTrmI. Increasing amounts of enzyme were incubated with 0.5μM 2-AP-mini-RNA in 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂ at 37°C for 5 min before monitoring the emission fluorescence spectrum (λ_exc = 320 nm).

Dynamics of RNA binding by _PabTrmI in the absence of methyl donor

To further investigate the dynamics of RNA binding to _PabTrmI, the pre-steady-state kinetics of 2-AP fluorescence increase at λ_ex = 320 nm after the rapid mixing of either 2AP57 or 2AP58 with an excess of free enzyme in the absence of SAM were monitored by stopped flow in fluorescence mode. When 1 μM 2AP57 or 2AP58 was mixed with 6 μM _PabTrmI, an increase in the fluorescence emission of the 2-AP-containing RNAs was observed within a second (Figure 4), followed by several slower processes. The kinetics of 2AP57 and 2AP58 binding by _PabTrmI were best fitted with three exponentials (Table 1). The binding of 2AP57 by _PabTrmI was slightly faster than that of 2AP58 (k₁ and k₃ of 2AP57 are, respectively, 1.4 and 2.5 times faster than those of 2AP58), in agreement with a better affinity of the enzyme for 2AP57.

Figure 4. — Base destacking kinetics of the 2-AP containing mini-RNAs by _PabTrmI in the absence of SAM at 37°C measured with a stopped-flow spectrofluorimeter. After the rapid mixing of the _PabTrmI (1 μM) from one syringe with either 2AP57 or 2AP58 (1 μM) from another syringe, the total fluorescence was recorded with a WG-360 cut-off filter after exciting at 320 nm. The experimental data points are shown as circles while the simulation curves are in solid and dashed lines for 2AP57 and 2AP58, respectively. The kinetics were best fitted with three exponentials.

Table 1. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 by _PabTrmI in the absence of cofactor.

	A₁	A₂	A₃	k₁	k₂	k₃
		(%)			(s⁻¹)
2AP57	43	39	18	1.8	0.34	0.05
2AP58	28	32	40	1.3	0.2	0.02

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Dynamics of RNA binding by _PabTrmI in the presence of SAM

To get further insights into the recognition mechanism of A₅₇ and A₅₈ under catalytic conditions, _PabTrmI was premixed with a saturated concentration of SAM, the methyl donor, before mixing with the 2-AP-mini-RNAs. Importantly, the addition of SAM to the 2-AP-mini-RNAs did not affect the fluorescence level of 2-AP (Supplementary Figure S4). The kinetics were recorded after excitation at 320 nm as aforementioned (Table 2 and Figure 5). Remarkably, not only the shapes of the kinetics curves were different from those observed in the absence of SAM but they also differed markedly between 2AP57 and 2AP58 (Figure 5A and B).

Table 2. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 and G57-2AP58-miniRNA by _PabTrmI in the presence of SAM or SAH.

	A₁	A₂	A₃	k₁	k₂	k₃
		(%)			(s⁻¹)
2AP57 + SAM	36	64	(100)*	7.62	1.1	(0.12)*
2AP57 + SAH	22	37	41	2.9	0.3	0.04
2AP58 + SAM	24	48	31	2.8	0.6	0.13
2AP58 + SAH	18	44	38	1.3	0.2	0.04
G57-2AP58 + SAM	34	36	30	2.5	0.3	0.04
G57-2AP58 + SAH	29	27	44	2.5	0.3	0.03

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()* this phase represents a decay of fluorescence corresponding to the return of the 2-AP to its canonical position in 2AP57 by _PabTrmI. This phase was observed only with 2AP57 in the presence of SAM. The kinetics were measured at 55°C.

Figure 5. — Base destacking kinetics of the 2-AP containing mini-RNAs by _PabTrmI in the presence of SAM (solid lines) and SAH (dashed lines) measured by stopped-flow spectrofluorimetry at 55°C. _PabTrmI (6 μM) and either SAM or SAH (1 mM) were pre-incubated for 10 min in one syringe, whereas the other syringe contained the 2-AP-mini-RNA with either SAM or SAH (1 mM). The reaction was started with the rapid mixing of both syringes and the total fluorescence was recorded with a WG-360 cut-off filter after exciting at 320 nm. (A) 2AP57. (B) 2AP58. (C) G57-2AP58-mini-RNA.

In the case of 2AP57, the kinetics were best fitted to three exponentials, a biphasic exponential increase of fluorescence during the first 5 s (7.62 s⁻¹ and 1.1 s⁻¹) being followed by a decrease with a rate constant of 0.12 s⁻¹ (Table 2 and Figure 5A).

In the case of 2AP58, the kinetics were also triphasic (rate constants of 2.8 s⁻¹, 0.6 s⁻¹ and 0.13 s⁻¹) but less complex, with a steady increase of fluorescence until a plateau was reached at ∼13 s (Table 2 and Figure 5B). The third kinetics phase was associated with a fluorescence increase, in contrast to 2AP57.

The fluorescence change of 2-AP in G57-2AP58-mini-RNA was also monitored under catalytic conditions, in the presence of SAM and enzyme (Figure 5C). _PabTrmI did not methylate G57-2AP58-mini-RNA (data not shown). Therefore, the use of this mini-RNA, in which the target adenine at position 57 is replaced by another purine that is not modified, enables to set aside the influence of the methylation reaction on the base(s) recognition mechanism. Despite the fact that the presence of the amino group of guanine could have an impact on the kinetics curves, these were very similar between G57-2AP58-mini-RNA and 2AP58. The RNA binding kinetics appeared to be only slightly slower and there was no observed decrease of fluorescence intensity when a guanine is present at position 57 rather than an adenine (Table 2).

Effect of SAH on RNA binding and base recognition by _PabTrmI

To investigate the effect of the SAH product on RNA binding, the protein was first incubated with an excess of SAH and then rapidly mixed with the various 2-AP-mini-RNAs in the stopped flow fluorimeter. The major phases of rate constants with 2AP57, 2AP58 or G57-2AP58-mini-RNA in the presence of SAH were very similar (Table 2 and Figure 5). Moreover, these kinetics were also similar to those observed with 2AP58 and G57-2AP58-mini-RNA in the presence of SAM, with no decrease of fluorescence.

Energy associated with the binding of RNA to _PabTrmI

To determine the activation energies associated with RNA binding, base recognition and methylation of A₅₇ and A₅₈ by _PabTrmI, the kinetics were recorded with 2AP57, 2AP58 or the G57-2AP58-mini-RNA in the presence of SAM at different temperatures (Supplementary Figure S5 and Supplementary Table S2). Raising the temperature enhanced the rate constants with both 2AP57 and 2AP58 without altering significantly the overall shape of the kinetics curves (Supplementary Figure S5). In the case of 2AP57, the overall amplitude of the signal diminished as the temperature increased (Supplementary Figure S5A), indicating a more pronounced temperature dependence for the last phase as evidenced by the larger activation energy (E_a3 ∼27.7 kcal mol⁻¹) (Supplementary Figure S6 and Table S2).

Structural model of _PabTrmI in complex with tRNA

We have recently solved the crystal structure of _PabTrmI in free form and in complex with either SAM or SAH cofactor (16). Unfortunately and despite numerous attempts, the tRNA/TrmI complex could not be crystallized. To understand the molecular basis for the RNA recognition and binding mode employed by _PabTrmI, we built a structural model of the enzyme/tRNA complex (Figure 6). The model was generated by rigid manual docking using the tetrameric structure of _PabTrmI in complex with four SAM molecules (PDB code 3MB5) and a full-length maturated yeast tRNA^Phe (PDB code 1EVV). Each monomer is organized into two domains, the N-terminal (Nt) domain and the catalytic C-terminal (Ct) domain containing the SAM binding pocket. Two pairs of monomers are associated into two dimers that combine to generate a stable tetramer. This oligomeric structure is conserved across evolution, as evidenced by several available crystal structures of _PabTrmI orthologs (17). Interestingly, two wide and large positively charged clefts are located on the sides of the tetramer (Figure 6 and Supplementary Figure S7). Two tRNA molecules can be accommodated in these putative RNA binding pockets without clashes. In the model, the T-stem loop and acceptor stem of the tRNAs contact the catalytic domains, the anticodon region being completely oriented outside the protein. In addition, in each RNA binding site, the D-loop of the tRNA interacts with the Nt-domain of one monomer, while the double-stranded acceptor and the T-stems contact the Nt-domain of the second monomer. In this configuration, the T-loop that contains nucleotides 57 and 58 lies in close proximity of SAM, whose methyl group is oriented toward the T-loop.

Figure 6. — Model of _PabTrmI in complex with tRNA. (A) Cartoon, (B) surface and (C) electrostatic representations of the enzyme/tRNA complex oriented in the same fashion. The different secondary structures of the tRNA are colored as follows: the T-psi stem loop in pale green, the D-stem loop in cyan, the anticodon stem loop in red, the variable loop in blue and the acceptor stem in salmon. The Nt and Ct domains of _PabTrmI are in violet and green, respectively. The SAM cofactor and the target adenines are represented in red. (D) 90° rotation view of (C).

The superposition of the _PabTrmI/tRNA model with the structures of the mono-site specific TrmI from T. thermophilus and A. aeolicus (_ttTrmI and _aaTrmI) shows that, although their catalytic domains align well, their Nt-domains are oriented differently compared to those of _PabTrmI (Supplementary Figure S8). Keeping the same orientation of the tRNA as in the _pabTrmI complex model provokes severe clashes of the tRNA with the Nt-domains of _ttTrmI and _aaTrmI. A reorientation of the tRNA molecules and/or conformational changes of the tRNA or proteins are necessary to prevent these clashes and properly fit the macromolecule substrate into the groves of the _PabTrmI homologs.

DISCUSSION

Our previous results indicated that the bi-specific _PabTrmI methylates A₅₈ in tRNAs containing the A₅₇A₅₈A₅₉ sequence only if A₅₇ has already been methylated (16). The present methylation assays indicate that the 29-mer P. abyssi mini-tRNA^Asp and mini-tRNA^Arg constitute good substrates of _PabTrmI as they are modified similarly to full-length tRNAs and thus they can be utilized to replace the target adenines by 2-AP. Interestingly, the enzyme did not methylate 2-AP, thereby discriminating between adenine, which contains an amino group at the C6 position, and 2-AP, in which this group is present at C2 instead of C6. Methylation kinetics under single-turnover conditions showed that A₅₈ is more efficiently methylated than A₅₇ when 2-AP is present at the adjacent position (57 and 58, respectively). The enzyme methylates quite efficiently A₅₈ in 2AP57 probably because the introduction of 2-AP at position 57 does not much interfere with the T-loop structure nor the correct placement of A₅₈ in the active site. In contrast, in 2AP58, the less efficient methylation of A57 may be due to the non-native reverse Hoogsteen base pair between U54 and 2-AP58 and/or steric hindrance caused by the NH₂ group at C2, which could hamper the optimal positioning of the nucleotides. _PabTrmI also methylated efficiently A₅₈ in a mini-RNA containing a guanine at position 57. Hence, methylation occurs at position 58 when position 57 contains a methylated adenine, an adenine derivative or guanine, whereas the methylation at position 57 strictly requires adenine 58 to proceed efficiently. This supports our previous conclusion that A57 is methylated before A58 in tRNAs containing the A₅₇A₅₈A₅₉ sequence (16).

The fluorescence properties of 2-AP enabled us to study the binding of RNA to _PabTrmI. The observed increase of the 2-AP fluorescence suggests that the binding of _PabTrmI to the 2AP-containing RNAs provokes base unstacking of the fluorescent probe itself and/or its immediate neighbors. Interestingly, this variation of fluorescence is in the same order of magnitude as that measured for placement of the target base in the active site of two RNA modifying enzymes (30–32). Accordingly, the model of _PabTrmI in complex with tRNA clearly underlines that the enzyme has to employ a base flipping mechanism to get access to both A₅₇ and A₅₈. The flipping of several bases in the vicinity of the target base by RNA modifying enzymes is a widely used mechanism, as reviewed by Li (21). The calculated apparent K_d values for 2AP57 and 2AP58 of 1.7 and 2.8 μM, respectively, indicate that both 2-AP-mini-RNAs are recognized by _PabTrmI. Although the enzyme exhibits a slightly higher affinity for 2AP57, in agreement with the slightly faster binding of 2AP57 by _PabTrmI compared to 2AP58, the position of 2-AP does not substantially alter RNA binding.

The binding kinetics of the 2-AP-containing mini-RNAs were then analyzed by stopped flow in the presence or absence of SAM or SAH. The rather complex multiphasic kinetics indicate that several molecular changes around the target bases occur, when _PabTrmI binds to the RNAs. Similar complex kinetics have been observed for adenine glycosylase MutY (33). Indeed, stopped-flow fluorescence analysis of MutY, using a duplex oligodeoxyribonucleotide containing a uracil-2-AP base pair, revealed that the extrusion of the targeted base was followed by slower protein isomerization steps that provided an additional stabilization of the flipped out adenine in the active site, thereby facilitating excision and base repair.

In the absence of SAM, the fluorescence increase was not followed by any decrease, suggesting that _PabTrmI recognizes both target bases in 2AP57 and 2AP58 and that 2-AP (and/or its neighbor nucleotides) may be stabilized in a destacked conformation. The enzyme is therefore able to recognize and bind RNA even in the absence of methyl donor. Remarkably, in the presence of SAM, a decrease of fluorescence occurred after the initial fluorescence increase only in the case of 2AP57. The first two increasing phases represent most likely the series of molecular events regarding the binding of the RNA and base recognition, while the last decreasing phase may be attributed to RNA dissociation after methylation of A₅₈. The absence of fluorescence decrease with 2AP58 could reflect the fact that 2-AP remains bound to the enzyme catalytic pocket or the return of 2-AP in the canonical position inside the tRNA is an extremely slow process. The similar kinetic constants shared by 2AP58 and G57-2PA58-mini-RNA, which does not undergo methylation, indicate that the observed signal of fluorescence likely originates from common events, i.e. RNA binding and base recognition in the vicinity of 2-AP. The kinetic curves for these mini-RNAs, compared with those for 2AP57, also suggest that the environment changes occurring around the 2-AP region are maintained as long as the methylation at position 58 is not achieved. Indeed, the methylation of A58 provides a gain in the stabilization energy of the U54-A58 or T54-A58 base-pairs of 6.8 kcal/mol (34), which agrees with the idea that methylation of A58 will trigger the dissociation of tRNA promoted by this high driving force. We thus propose that the RNA binding dynamics are orchestrated by the methylation of adenine 58. Such a crucial role of the methylation of A₅₈ is in agreement with m¹A₅₈ being universally conserved in tRNAs.

The kinetics in the presence of SAH for 2AP57, 2AP58 or G57-2AP58-mini-RNA were similar to those of 2AP58 or G57-2AP58-mini-RNA in the presence of SAM. This suggests that the enzyme is capable of binding these mini-RNAs and induce a destacking of the 2-AP and/or its immediate neighbors at a similar rate, independently of the presence of SAH or SAM. Therefore, the specific recognition of the RNA by _PabTrmI does not appear to rely on the presence of the SAM cofactor.

Finally, the temperature dependence of the kinetic constants was measured and the activation energies corresponding to the three main steps were determined by Arrhenius plots. In the case of 2AP57, the maximum of fluorescence intensity diminished as the temperature increased, indicating that the rate of the last phase assigned to the dissociation of the RNA is enhanced more than that of the first phases assigned to the recognition process, which results in a large E_a3 value. The large E_a1 E_a2 and E_a3 activation energies determined for 2AP57 may reflect the fact that the corresponding steps include the methylation of A₅₈ because the E_a values for 2AP58 and the G57-2AP58-mini-RNA, in which methylation of A₅₈ does not occur, are similar and relatively weak compared to those observed with 2AP57. Hence, the recognition of A₅₈ together with its methylation and the flipping back of m¹A₅₈ into the RNA may contribute significantly to the high activation energies observed with 2AP57.

Although the _PabTrmI tetramer binds four SAM molecules, our model of its complex with tRNA underscores the symmetric binding of two tRNA molecules and the use of only two SAM molecules over four for catalysis. The binding of two tRNA molecules is in agreement with the MS experiments carried out with _ttTrmI (19). Our model shows that the protein interacts mainly with the T-psi stem loop and the acceptor stem of the tRNA, which is in agreement with our activity test showing that a mini-RNA substrate containing only these regions is recognized by _PabTrmI and methylated. The model also illustrates that the flipping of both A₅₇ and A₅₈ is mandatory to bring both targets at a suitable distance for methyl transfer. In particular, flexible loops of the Nt-domain that are in close contact with the T-loop could be determinant to untighten the tertiary interactions at the elbow of the tRNA (intersecting region between the D- and T-loops) and promote base flipping. Except base flipping, no dramatic conformational change of the tRNA appears necessary for the reaction to occur, as observed for most RNA-modifying enzymes. In particular, T. thermophilus dihydrouridine synthase, which modifies U20 in the D-loop of tRNA, does not require extensive conformational changes in the tRNA to access its target base because the canonical D-loop/T-loop interaction was maintained in the crystal structure of the complex (35). The larger RNA binding pocket of _PabTrmI compared to that of other mono-site-specific TrmIs (17) may enable much more flexibility in the binding of the tRNA and would allow the enzyme to position A57 or A58 for methylation without serious steric hindrance. In addition, the rather large solvent accessibility of the SAM binding pocket, even in the presence of tRNA, agrees with the fact that the first SAH molecule could dissociate after methylation of the first adenine and be replaced by the second SAM molecule without provoking tRNA dissociation, thereby enabling the consecutive methylation of the second adenine. This scenario is supported by the comparison of the crystal structures of _PabTrmI alone, in complex with SAH or SAM, which does not highlight noticeable conformational changes (16). A general mechanism for the double methylation of tRNA by _PabTrmI can thus be proposed (Figure 7).

Figure 7. — Proposed mechanism for the methylation of A₅₇ and A₅₈ by _PabTrmI. In the presence of SAM (red circle), the tetrameric _PabTrmI binds two tRNA molecules, recognizing specifically the T-psi stem-loop containing the region to be modified. Then, the enzyme destacks and flips out A₅₇ alone or both A₅₇ and A₅₈ from the tRNA. The proximity of A₅₇ to the SAM binding pocket enables its methylation first. The release of the first SAH molecule (blue circle), which does not promote the release of the tRNA from the enzyme, is followed by the binding of the second SAM molecule and leads to the methylation of A₅₈. The production of m¹A₅₈ triggers the release of tRNA and, consequently, the enzyme, liberated from the products, becomes ready for another turnover.

Multi-site RNA modifying enzymes may share a similar sequential dynamical process and the same method that uses oligonucleotides containing 2-AP at the target positions may be implemented for the investigation of the RNA conformational changes occurring during catalysis by these enzymes.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

SUPPLEMENTARY DATA

supp_42_18_11697__index.html^{(771B, html)}

FUNDING

CNRS. Funding for open access charge: French State Program ‘Investissements d'Avenir’ [LABEX DYNAMO ANR-11-LABX-0011].

Conflict of interest statement. None declared.

REFERENCES

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Supplementary Materials

SUPPLEMENTARY DATA

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[B1] 1.Gustilo E.M., Vendeix F.A., Agris P.F. tRNA's modifications bring order to gene expression. Curr. Opin. Microbiol. 2008;11:134–140. doi: 10.1016/j.mib.2008.02.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Motorin Y., Helm M. tRNA stabilization by modified nucleotides. Biochemistry. 2010;49:4934–4944. doi: 10.1021/bi100408z. [DOI] [PubMed] [Google Scholar]

[B3] 3.El Yacoubi B., Bailly M., de Crecy-Lagard V. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu. Rev. Genet. 2012;46:69–95. doi: 10.1146/annurev-genet-110711-155641. [DOI] [PubMed] [Google Scholar]

[B4] 4.Agris P.F. Decoding the genome: a modified view. Nucleic Acids Res. 2004;32:223–238. doi: 10.1093/nar/gkh185. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Awai T., Kimura S., Tomikawa C., Ochi A., Ihsanawati, Bessho Y., Yokoyama S., Ohno S., Nishikawa K., Yokogawa T., et al. Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA. J. Biol. Chem. 2009;284:20467–20478. doi: 10.1074/jbc.M109.020024. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Awai T., Ochi A., Ihsanawati, Sengoku T., Hirata A., Bessho Y., Yokoyama S., Hori H. Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure. J. Biol. Chem. 2011;286:35236–35246. doi: 10.1074/jbc.M111.253641. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Hur S., Stroud R.M. How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA. Mol. Cell. 2007;26:189–203. doi: 10.1016/j.molcel.2007.02.027. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Poldermans B., Roza L., Van Knippenberg P.H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3′ end of 16 S ribosomal RNA of Escherichia coli. III. Purification and properties of the methylating enzyme and methylase-30 S interactions. J. Biol. Chem. 1979;254:9094–9100. [PubMed] [Google Scholar]

[B9] 9.Cunningham P.R., Weitzmann C.J., Nurse K., Masurel R., Van Knippenberg P.H., Ofengand J. Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase. Biochim. Biophys. Acta. 1990;1050:18–26. doi: 10.1016/0167-4781(90)90135-o. [DOI] [PubMed] [Google Scholar]

[B10] 10.Tu C., Tropea J.E., Austin B.P., Court D.L., Waugh D.S., Ji X. Structural basis for binding of RNA and cofactor by a KsgA methyltransferase. Structure. 2009;17:374–385. doi: 10.1016/j.str.2009.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Roovers M., Wouters J., Bujnicki J.M., Tricot C., Stalon V., Grosjean H., Droogmans L. A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase. Nucleic Acids Res. 2004;32:465–476. doi: 10.1093/nar/gkh191. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Anderson J., Phan L., Cuesta R., Carlson B.A., Pak M., Asano K., Bjork G.R., Tamame M., Hinnebusch A.G. The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA. Genes Dev. 1998;12:3650–3662. doi: 10.1101/gad.12.23.3650. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Droogmans L., Roovers M., Bujnicki J.M., Tricot C., Hartsch T., Stalon V., Grosjean H. Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures. Nucleic Acids Res. 2003;31:2148–2156. doi: 10.1093/nar/gkg314. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Grosjean H., Constantinesco F., Foiret D., Benachenhou N. A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA. Nucleic Acids Res. 1995;23:4312–4319. doi: 10.1093/nar/23.21.4312. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Constantinesco F., Motorin Y., Grosjean H. Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro. Nucleic Acids Res. 1999;27:1308–1315. doi: 10.1093/nar/27.5.1308. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Guelorget A., Roovers M., Guérineau V., Barbey C., Li X., Golinelli-Pimpaneau B. Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase. Nucleic Acids Res. 2010;38:6206–6218. doi: 10.1093/nar/gkq381. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Guelorget A., Barraud P., Tisné C., Golinelli-Pimpaneau B. Structural comparison of tRNA m(1)A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions. BMC Struct. Biol. 2011;11:48. doi: 10.1186/1472-6807-11-48. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Kuratani M., Yanagisawa T., Ishii R., Matsuno M., Si S.Y., Katsura K., Ushikoshi-Nakayama R., Shibata R., Shirouzu M., Bessho Y., et al. Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine. J. Struct. Funct. Genomics. 2014;15:173–180. doi: 10.1007/s10969-014-9183-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

Djemel Hamdane

Amandine Guelorget

Vincent Guérineau

Béatrice Golinelli-Pimpaneau

Abstract

INTRODUCTION

MATERIALS AND METHODS

Material

Purification of PabTrmI

Mass spectrometry analysis

Steady-state fluorescence assay

Stopped-flow kinetics

RESULTS

PabTrmI recognizes and methylates both A57 and A58 in mini-tRNAAsp containing the A57A58A59U60 sequence

Figure 1.

PabTrmI recognizes and methylates only A58 in mini-tRNAArg (TCT) containing the G57A58A59U60 sequence

The 2-AP replacing the target adenines in a mini-RNA substrate is not methylated by PabTrmI

The methylation of A58 is not impaired by 2-AP at the adjacent position 57

Figure 2.

Interaction of 2-AP-containing mini-RNAs with PabTrmI

Figure 3.

Dynamics of RNA binding by PabTrmI in the absence of methyl donor

Figure 4.

Table 1. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 by PabTrmI in the absence of cofactor.

Dynamics of RNA binding by PabTrmI in the presence of SAM

Table 2. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 and G57-2AP58-miniRNA by PabTrmI in the presence of SAM or SAH.

Figure 5.

Effect of SAH on RNA binding and base recognition by PabTrmI

Energy associated with the binding of RNA to PabTrmI

Structural model of PabTrmI in complex with tRNA

Figure 6.

DISCUSSION

Figure 7.

SUPPLEMENTARY DATA

FUNDING

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Purification of _PabTrmI

_PabTrmI recognizes and methylates both A57 and A58 in mini-tRNA^Asp containing the A₅₇A₅₈A₅₉U₆₀ sequence

_PabTrmI recognizes and methylates only A58 in mini-tRNA^Arg (TCT) containing the G₅₇A₅₈A₅₉U₆₀ sequence

The 2-AP replacing the target adenines in a mini-RNA substrate is not methylated by _PabTrmI

The methylation of A₅₈ is not impaired by 2-AP at the adjacent position 57

Interaction of 2-AP-containing mini-RNAs with _PabTrmI

Dynamics of RNA binding by _PabTrmI in the absence of methyl donor

Table 1. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 by _PabTrmI in the absence of cofactor.

Dynamics of RNA binding by _PabTrmI in the presence of SAM

Table 2. Amplitudes and observed rate constants for the base destacking kinetics of 2AP57, 2AP58 and G57-2AP58-miniRNA by _PabTrmI in the presence of SAM or SAH.

Effect of SAH on RNA binding and base recognition by _PabTrmI

Energy associated with the binding of RNA to _PabTrmI

Structural model of _PabTrmI in complex with tRNA