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Biochem. J. (2013) 453, 455–465 (Printed in Great Britain) 455 doi:10.1042/BJ20130294 Multilevel functional and structural defects induced by two pathogenic mitochondrial tRNA mutations Meng WANG*†, Xiao-Long ZHOU*, Ru-Juan LIU*, Zhi-Peng FANG*, Mi ZHOU*, Gilbert ERIANI†1 and En-Duo WANG*1 *Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China, and †Architecture et Réactivité de l’ARN, Université de Strasbourg, Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France Point mutations in hmtRNAs (human mitochondrial tRNAs) can cause various disorders, such as CPEO (chronic progressive external ophthalmoplegia) and MM (mitochondrial myopathy). Mitochondrial tRNALeu , especially the UUR codon isoacceptor, is recognized as a hot spot for pathogenic mtDNA point mutations. Thus far, 40 mutations have been reported in hmtRNAsLeu . In the present paper, we describe the wide range of effects of two substitutions found in the TC arms of two hmtRNAsLeu isoacceptors. The G52A substitution, corresponding to the pathogenic G12315A mutation in tRNALeu (CUN), and G3283A in tRNALeu (UUR) exhibited structural changes in the outer corner of the tRNA shape as shown by RNase probing. These mutations also induced reductions in aminoacylation, 3 -end processing and base modification processes. The main effects of the A57G substitution, corresponding to mutations A12320G in tRNALeu (CUN) and A3288G in tRNALeu (UUR), were observed on the aminoacylation activity and binding to hmEF-Tu (human mitochondrial elongation factor Tu). These observations suggest that the wide range of effects may amplify the deleterious impact on mitochondrial protein synthesis in vivo. The findings also emphasize that an exact understanding of tRNA dysfunction is critical for the future development of therapies for mitochondrial diseases. INTRODUCTION tRNA. The enzyme protects the acceptor stem, D-stem/loop and anticodon stem/loop [12]. Therefore we screened the aminoacylation properties of all known pathogenic mutants of hmtRNALeu and identified two mutations of the T-stem/loop, G52A and A57G, that drastically decreased the aminoacylation capacity. The pathogenic mutation G52A was identified previously in both tRNALeu (CUN) (G12315A gene mutation) and in tRNALeu (UUR) (G3283A gene mutation). This mutation has been found to cause CPEO (chronic progressive external ophtalmoplegia) and/or encephalomyopathies in patients [13– 15]. The A57G mutation was also isolated in both tRNALeu isoacceptors. It corresponds to the pathological A12320G mutation in tRNALeu (CUN) and A3288G in tRNALeu (UUR), and results in limb and respiratory muscle weakness and degeneration [16,17]. In the present study, we examined the effects of the two mutations mentioned above on the structure and function of the two isoacceptors of hmtRNALeu , including conformational changes, the ability to be aminoacylated by hmLeuRS, processed by tRNA nucleotidyltransferase, modified by m1 G37 -methyltransferase or prenyltransferase, and the binding capacity of aminoacylated tRNALeu to hmEF-Tu [human mitochondrial EF-Tu (elongation factor thermo unstable)]. Systematic investigations of the structure and function of hmtRNAs can help to elucidate their pathogenic effects, provide information on the treatment of mitochondrial diseases, and potentially facilitate the development of diagnostic tools and therapies for these diseases, such as import of mitochondrially targeted functional aa-tRNA (aminoacyl tRNA) synthetase [18] or tRNAs [19]. Mitochondria are eukaryotic organelles that generate most of the energy in the cell by OXPHOS (oxidative phosphorylation). Human mitochondria contain a compact circular genome that is 16 569 bp in length, encoding 13 essential subunits of the inner membrane complex responsible for OXPHOS, two rRNAs and 22 species of hmtRNAs (human mitochondrial tRNAs) [1,2]. The primary and secondary structures of hmtRNAs differ significantly from those of canonical bacterial and cytoplasmic tRNAs, and tRNAs in human mitochondria are less thermodynamically stable as they generally contain higher numbers of mismatched and A/U base pairs [3]. hmtRNAs are highly susceptible to point mutations, which are the primary cause of the mitochondrial dysfunctions associated with a wide range of pathologies. During the last two decades, an increasing number of single nucleotide substitutions within the hmtRNA genes have been linked to a variety of diseases showing pleiotropic effects. More than 220 mutations have been associated with diseases in the 22 genes of hmtRNAs and 40 in hmtRNAsLeu [4–8]. Usually, these point mutations impair one or several steps of tRNA maturation as well as protein biosynthesis, including 5 - and 3 -end processing of precursor tRNA, post-transcriptional modification of bases, structural stability, aminoacylation, and formation of tRNA–ribosomal complexes [9–11]. Two different isoaccepting tRNAsLeu are found in human mitochondria, hmtRNALeu (CUN) and hmtRNALeu (UUR). It was reported that hmLeuRS (human mitochondrial leucyl tRNA synthetase) recognizes A14 and A73 as critical nucleotides in Key words: CCA-adding modification, elongation factor Tu (EF-Tu), mitochondria, pathogenicity, tRNA. Abbreviations used: aa-tRNA, aminoacyl tRNA; AdoMet, S -adenosylmethionine; CPEO, chronic progressive external ophthalmoplegia; DMAPP, dimethylallyl pyrophosphate; EF-Tu, elongation factor thermo unstable; hmEF-Tu, human mitochondrial EF-Tu; hmLeuRS, human mitochondrial leucyl tRNA synthetase; hmTNT, human mitochondrial tRNA nucleotidyltransferase; hmtRNA, human mitochondrial tRNA; MiaA, tRNA prenyltransferase; OXPHOS, oxidative phosphorylation; Trm5, tRNA-(N1 G37 ) methyltransferase; TrmD, tRNA (guanine-N1 -)-methyltransferase. 1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2013 Biochemical Society 456 M. Wang and others EXPERIMENTAL Enzyme purification and tRNA preparation All chemicals were purchased from Sigma–Aldrich, except when otherwise noted. T4 polynucleotide kinase, T4 DNA ligase, restriction endonucleases, RNase V1, S1, T1 and RNasin inhibitor were obtained from Fermentas. [3 H]DMAPP (dimethylallyl pyrophosphate) was obtained from BIOTREND Chemikalien. Other radioactive amino acids and ATP were purchased from PerkinElmer. Stains-All dye was purchased from Santa Cruz Biotechnology. Nitrocellulose membranes (0.22 μm) were obtained from Merck Millipore. T7 RNA polymerase was purified from Escherichia coli overproducing strains as described previously [20]. In vitro transcription of full-length or 3 -truncated tRNAs without a CCA terminus was performed as described previously [11]. The tRNA concentration was determined by UV absorbance at 260 nm, and the molar absorption coefficient was calculated according to the sequence of each tRNA [21]. UV melting studies tRNA variants (0.7 μM) were diluted in 50 mM potassium phosphate buffer (pH 7.0), with 100 mM NaCl and 0.1 mM EDTA. tRNAs were first heat-denatured at 95 ◦ C for 3 min and chilled at 0 ◦ C before the thermal denaturation experiment. Absorbance against temperature melting curves were measured at 260 nm with a heating rate of 0.2 ◦ C/min from 20 to 90 ◦ C on a UVIKON-XL spectrometer (SECOMAM) equipped with a Peltier thermostated cell holder. UV melting curves and their derivatives were smoothed with a Fourier-based filtering procedure programmed with Mathematica (Wolfram). Nuclease probing of tRNA structure tRNA transcripts were 5 -end radiolabelled using T4 polynucleotide kinase and [γ -32 P]ATP [22]. The 3 -end labelling of the tRNA transcripts was achieved using tRNA nucleotidyl transferase and [α-32 P]ATP [23]. Radioactive transcripts were further purified by denaturing 10 % PAGE and eluted overnight at 4 ◦ C in buffer containing 10 mM Tris/HCl (pH 7.5), 0.3 M NaCl and 0.5 mM EDTA. Before use, transcripts were folded in water by incubation at 80 ◦ C for 2 min, slow cooling to 35 ◦ C and kept on ice. 32 P-labelled tRNAs (50 000 c.p.m., ∼ 2 pmoles) were cleaved with various nucleases at 20 ◦ C for 10 min in 5 μl of 50 mM Tris/HCl (pH 7.6), 10 mM MgCl2 , 25 mM KCl, 50 ng/μl total yeast tRNA, and RNase T1 (0.125, 0.25 or 0.5 units), RNase V1 (0.001, 0.002 or 0.004 units) and nuclease S1 (1.67, 3.33 or 6.67 units). Reactions were stopped by adding 6 μl of stop mix [0.6 M sodium acetate (pH 5.0), 3 mM EDTA and 0.1 μg/μl total yeast tRNA], and samples were fractionated by 10 % denaturing PAGE [24]. RNase T1 ladders were made as described previously [25]. Alkaline ladders were obtained by incubation of the labelled transcript with 1 μg of yeast bulk tRNA for 10 min at 80 ◦ C in a buffer containing 80 mM Na2 CO3 /NaHCO3 (pH 9.0). Measurement of equilibrium dissociation constants for tRNA by filter-binding assays HmLeuRS was purified in our laboratory as described previously [26]. hmLeuRS–[32 P]tRNALeu complex formation was monitored using the nitrocellulose filter-binding method [27]. Nitrocellulose membranes (0.22 μm) were pre-soaked in washing buffer [50 mM potassium phosphate (pH 5.5) and 50 mM MgCl2 ] for at least c The Authors Journal compilation c 2013 Biochemical Society 10 min before use. The 32 P-labelled hmtRNALeu (6500 c.p.m., ∼ 2.5 pmoles) was incubated in a 50 μl of reaction volume in the presence of hmLeuRS at various concentrations for 15–30 min at 0 ◦ C in a buffer containing 50 mM Hepes/KOH (pH 6.8), 30 mM KCl and 12 mM MgCl2 . The samples were then applied and filtered through the nitrocellulose membrane. The filters were washed with 0.3 ml of washing buffer, dried and the radioactivity was counted. Data were analysed using GraphPad Prism software. Determination of kinetic parameters for the addition of CCA hmTNT (human mitochondrial tRNA nucleotidyltransferase), also known as CCA-adding enzyme, was purified as described previously [28]. CCA addition was monitored at 37 ◦ C in reaction mixtures containing 50 mM Tris/HCl (pH 8.5), 10 mM MgCl2 , 100 mM KCl, 0.1 mM CTP, 0.1 mM [α-32 P]ATP, 1 mM DTT, 0.05 % BSA, 5 μM 3 -truncated tRNAs without CCA terminus and 1 μM recombinant hmTNT. For determination of kinetic parameters, 3 -truncated tRNAs were incubated under the indicated conditions for 15 min at final concentrations ranging from 0.1 μM to 10 μM. The resulting reaction products were separated by denaturing 10 % PAGE, which were exposed to an imaging plate. Signal intensities were quantified and used to determine kinetic parameters [29]. In vitro base modification assays Human Trm5 [tRNA-(N1 G37 ) methyltransferase] and E. coli TrmD [tRNA (guanine-N1 -)-methyltransferase], a homologue of Trm5, were purified as described [30,31]. Wild-type tRNA or G52A and A57G mutants (5 μM) were incubated at 37 ◦ C in a reaction mix containing 50 mM Tris/HCl (pH 8.0), 24 mM NH4 Cl, 6 mM MgCl2 , 1 mM DTE (dithioerythritol), 0.1 mM EDTA, 40 μM [3 H]AdoMet (S-adenosylmethionine), and 5 μM Trm5 or TrmD. At time intervals ranging between 5 and 25 min, aliquots of 10 μl were removed, absorbed on paper discs and precipitated in trichloroacetic acid according to the protocol used for measuring aminoacylation reactions [32]. E. coli MiaA (tRNA prenyltransferase; EC 2.5.1.8), commonly named tRNA isopentenyl transferase, was expressed as an Nterminal His6 tag fusion protein. MiaA protein was purified following a previously described protocol used for His-tagged hmLeuRS [11]. Wild-type tRNALeu (UUR) and the corresponding G52A or A57G mutants (5 μM) were incubated at 37 ◦ C with 1 μM of MiaA enzyme in 50 mM Tris/HCl (pH 7.5), 3.5 mM MgCl2 , 5 mM 2-mercaptoethanol, 1 mg/ml BSA and 25 μM [3 H]DMAPP. At various time intervals, 10 μl aliquots were removed, absorbed on paper and processed as in the Trm5 and TrmD assays. Aminoacylation and hmEF-Tu protection assays The accepting activity of the tRNA transcripts and the time course of tRNA aminoacylation were assessed as described previously [11]. The aminoacylation reaction mixture contained 50 mM Hepes (pH 7.6), 25 mM KCl, 10 mM MgCl2 , 2.5 mM ATP, 1 mM spermidine, 100 μg/ml BSA, 40 μM L-[14 C]leucine, 0.3 μM hmLeuRS and 5 μM hmtRNALeu transcript, for times ranging between 5 and 25 min. Ternary complex formation (hmEF-Tu–GTP–Leu-tRNALeu ) was assayed by examining the ability of hmEF-Tu to protect [14 C]Leu–tRNALeu from spontaneous deacylation in water. hmEFTu was purified as described previously [33], and the procedure of protection of Leu–tRNALeu by hmEF-Tu also was derived Functional and structural defects in tRNA mutants Figure 1 457 Pathogenic hmtRNALeu mutations examined in the present study and their basic aminoacylation properties (A and B) Cloverleaf structures of the two human mitochondrial tRNALeu with associated mutations. Broken lines represent tertiary base pairs typically found in tRNAs. (C and D) Time curves of aminoacylation of wild-type (WT) and mutated hmtRNAsLeu by hmLeuRS. Results are reported as averages of three assays and error bars indicate S.D. from a previously published protocol [34]. Preformed [14 C]Leu– tRNALeu (3000 c.p.m., 1 μM) was incubated with purified hmEFTu (5 μM) at 37 ◦ C in 75 mM Tris/HCl (pH 7.6), 75 mM NH4 Cl, 15 mM MgCl2 , 7.5 mM DTT, 60 μg/ml BSA, 1 mM GTP and 2.4 mM PEP (phosphoenolpyruvate)/0.1 m-units PK (pyruvate kinase), for times ranging between 5 and 30 min. At various times, 10 μl aliquots were removed, absorbed on paper disks and precipitated by 5 % trichloroacetic acid [32]. RESULTS four mutated tRNAs by filter-binding assays on nitrocellulose membranes. The K d value of wild-type hmtRNALeu (CUN) was 1.65 μM, and those from mutants G12315A and A12320G were 1.84 and 2.43 μM respectively. For the second isoacceptor hmtRNALeu (UUR), the K d values were 1.47, 1.68 and 1.88 μM for the wild-type and G3283A and A3288G mutations respectively (Supplementary Figure S1 at http://www.biochemj.org/bj/453/ bj4530455add.htm). Thus the mutations did not induce a severe loss in affinity for the synthetase. The mutated tRNAs may harbour changes in the tertiary structure resulting from the D- and T-loop interactions, which has previously been shown to be responsible for pathogenic defects in hmtRNALeu [11,35]. Selection of two mutations that preclude efficient charging of hmtRNAsLeu The two isoacceptors hmtRNALeu (UUR) and hmtRNALeu (CUN) have a classical cloverleaf structure and a short variable arm in contrast to bacterial tRNALeu , which displays the long variable arm characteristic of class II tRNAs (Figures 1A and 1B). In a preliminary study, we explored the aminoacylation properties of 40 transcripts carrying mutations identified previously as well as two wild-type tRNAsLeu . Several transcripts bearing mutations in the T-arm showed significant decreases in aminoacylation when compared with the corresponding wildtype isoacceptor (results not shown). We focused our interest on two similar mutations in the T-arm at positions G52 and A57 of both hmtRNALeu (UUR) and hmtRNALeu (CUN). Plateaus of tRNA charging reached only 30 % and charging rates were considerably reduced compared with the wild-type transcripts (Figures 1C and 1D). As aminoacylation efficiency may also result from improper tRNA binding, we measured the dissociation constants of the Melting effects of mutations To further explore the putative effects of mutations on the global folding of transcripts, we performed PAGE analysis under native and denaturing conditions. On a denaturing 8 M urea gel, transcripts of hmtRNALeu (CUN) (74 nt), E. coli tRNAVal (77 nt) and hmtRNALeu (UUR) (78 nt) migrated according to their sizes. Significant differences were found during the migration in native conditions. The two G52A mutants ran more slowly than the four others (Figure 2A). Altogether, the data showed that the conformation of the mutated hmtRNAsLeu was modified compared with the corresponding wild-type tRNAsLeu . Consequently, we studied the UV melting curves of the four mutants and compared them with those of the corresponding wild-type transcripts (Supplementary Figure S2 at http:// www.biochemj.org/bj/453/bj4530455add.htm). The melting temperatures (T m ) were determined by calculating the derivatives c The Authors Journal compilation c 2013 Biochemical Society 458 M. Wang and others Conformational changes induced by pathogenic mutations in hmtRNALeu Figure 2 Analysis of conformations of wild-type hmtRNAsLeu and pathogenic mutants (A) Native and denaturing PAGE analysis of tRNA transcripts. A total of 2 μg of each transcript was loaded, and the gels were stained by Stains-All. Fully modified E. coli tRNAVal was used as a control. (B and C) Melting profiles were measured at 260 nm with a heating rate of 0.2 ◦ C/min from 20 to 90 ◦ C. First derivative (dA/dT) against temperature curves are shown to highlight the T m value transitions. WT, wild-type. of the absorbance against a temperature curve. The melting profiles of the transcripts were multiphasic as expected from RNAs exhibiting different levels of folding with secondary and tertiary structures. In general, a first transition was observed upon the breakdown of the tertiary structure of the tRNA at 39.1 ◦ C and 36.6 ◦ C for hmtRNALeu (CUN) and hmtRNALeu (UUR) respectively (Figures 2B and 2C). At higher temperatures additional transitions were observed resulting from the melting of the arms of the tRNA [36]. Remarkably, the mutant curves did not superimpose perfectly with the wild-type ones. For hmtRNALeu (UUR) mutants, differences were mainly found in transitions at high temperatures, whereas the first transition at low temperature remained essentially unchanged (differences in T m value were within 1.5 ◦ C) compared with the wild-type. These results suggested that the mutations may have affected the structure of the arms of the tRNA. Only the G3283A mutant (G52A) of hmtRNALeu (UUR) showed variations of melting transitions, with the T m value of the first transition obviously decreased from 39.1 ◦ C to 35 ◦ C. These changes suggest that the global tRNA folding of the G52A mutant was less stable at high temperatures. c The Authors Journal compilation c 2013 Biochemical Society Structures of the four mutated tRNAs and corresponding wildtype tRNAs were probed by treatment with RNases T1, V1 and S1. RNase T1 cuts preferentially after unpaired glycine residues and RNase S1 after unpaired residues. RNase V1 cuts in double-stranded sequences or higher-order structures. The tRNA structures were probed after labelling at their 5 - or 3 -ends. Three concentrations of nucleases were used and the cut positions were identified with a G-ladder (guanosine monophosphate ladder) obtained by RNase T1 digestion under denaturing conditions and an alkaline ladder obtained by hydroxyl cleavage at pH 9.0. Probing of the wild-type hmtRNALeu (CUN) confirmed the typical cloverleaf structure. RNase V1 exhibited a clear preference for the anticodon stem and RNase S1 for the nucleotides of the variable region, whereas the RNase T1 cleavage sites mainly were found in the D-loop and anticodon-loop (Figures 3A and 3B). The structure of the G52A mutant was obviously changed in the variable loop and T-arm as shown by the main changes in the cleavage patterns of the three RNases. RNase S1 cuts revealed that the variable loop was much more accessible. Similarly, doublestrand cuts (V1) were reduced on one side of the anticodon stem and increased on the other side, whereas new cuts appeared in the T-arm and unexpectedly in the T-loop. New RNase T1 cuts appeared in the T-loop and previous cuts were amplified in the anticodon loop. Altogether, these data suggested a wide change in the tertiary structure of the tRNA with the consequence of the variable loop and the eight following residues becoming more sensitive to RNase. The presence of new RNase V1 cuts also indicated a change in the higher order structure of the tRNA with establishment of new tertiary interactions. The insertion of the G52A mutation in the T-stem, which already contained three AU base pairs, decreased its stability and changed the tRNA conformation (Figure 3C). The structure of the A57G mutant was probed under the same conditions. No changes could be detected in the digestion profile of the mutant compared with that of the wild-type, except at the G57 mutation site where a new T1 cut appeared. Therefore the probing analysis suggested that the A57G mutation did not change the tRNA structure significantly, at least in the experimental conditions tested in the present study (Figure 3C). Enzymatic probing of the wild-type hmtRNALeu (UUR) structure revealed a different cleavage pattern compared with that of hmtRNALeu (CUN). The strong V1 cuts observed previously in the anticodon stem of hmtRNALeu (CUN) were not seen in hmtRNALeu (UUR); instead, reactivity was strong with RNase S1 in the stem and loop, suggesting that the anticodon loop of the tRNA containing four AT base pairs and ends with an AC pair could be easily melted. In general, RNase V1 cuts were very weak and we were unable to obtain cuts with stronger intensity despite several attempts to clarify the result. Nevertheless, we could detect several weak signals that differed between the different tRNAs (Figures 4A and 4B). Compared with the wild-type, the G52A mutant exhibited changes in the cleavage patterns as the T-stem became more accessible to RNase T1 and the variable loop to RNase S1 cuts. These results may be explained by the difference in the sequence of the T-loop which does not allow the slippage of the arm as proposed for the isoacceptor hmtRNALeu (CUN). Nevertheless, the probing analysis suggested that the accessibility of this region of the tRNA to RNases was significantly changed and that the conformation of the tRNA was modified by the mutation (Figure 4C). The A57G mutant showed basically the same cleavage pattern as that of the wild-type tRNA with one additional T1 cut at the mutation site (Figure 4C). Functional and structural defects in tRNA mutants Figure 3 459 Enzymatic probing of the three tRNALeu (CUN) and effect of mutations on tRNA secondary structures Enzymatic probing analysis. Probing was performed by various RNase T1, V1 and S1 concentrations. tRNAs were 5 radiolabelled with T4 polynucleotide kinase (A) or 3 radiolabelled using E. coli tRNA nucleotidyltransferase (B). Lane T1, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. (C) Structure analysis by enzymatic probing. tRNA regions where significant reactivity changes occurred are highlighted in grey. Taken together, these probing experiments showed that G52A mutations induced significant conformational changes in the tRNA structure of the two tRNAs, whereas the A57G mutations did not modify the cleavage patterns. Mutations in the T-stem of hmtRNAsLeu interfere with tRNA 3 -end processing In mitochondria, tRNAs are transcribed as long polycistronic RNAs containing neither introns nor CCA 3 -ends. In order to become functional, tRNA precursors must be processed at both the 5 - and 3 -end to remove redundant sequences and then maturated by addition of the CCA sequence [2]. In the present study, we examined whether G52A or A57G mutations could also impact on the CCA addition process. Therefore we monitored CCA synthesis on tRNA precursors obtained by in vitro run-off transcription of tRNA gene templates deprived of the CCA sequence. It has been shown that T7 RNA transcripts devoid of post-transcriptional modifications are proper substrates for end-maturation studies since these processes occur before modification of the bases [37]. When tested as substrates for CCA addition, G52A mutants of both hmtRNALeu (CUN) and hmtRNALeu (UUR) showed consequent decreases in their catalytic efficiency of 11.9- and 10.7-fold respectively (Figures 5A and 5B, and Table 1). The mutation mainly impacted on the observed catalytic rate in hmtRNALeu (CUN), whereas it had a strong impact on the Michaelis constant in hmtRNALeu (UUR). This finding suggested that the affinity of tRNA nucleotidyl transferase for the mutant tRNA was reduced compared with the wild-type isoacceptor (UUR), whereas the chemical step of the reaction seemed to be affected in the isoacceptor (CUN). In contrast, the pathogenic mutation A57G produced a poor effect on CCA addition and kinetic behaviours were comparable with the wildtype transcript (Table 1). Crystal structures of tRNA nucleotidyl transferases have revealed that these enzymes bind the upper part of the L-shaped tRNA, consisting of the coaxial alignment of the acceptorand T-stems (Figure 5C). These interactions include non-basespecific hydrogen bonding and electrostatic interactions with the c The Authors Journal compilation c 2013 Biochemical Society 460 Figure 4 M. Wang and others Enzymatic probing of the three tRNAsLeu (UUR) and effect of mutations on tRNA secondary structures Enzymatic probing analysis. Probing was performed by various RNase T1, V1 and S1 concentrations. tRNAs were 5 radiolabelled with T4 polynucleotide kinase (A) or 3 radiolabelled using E. coli tRNA nucleotidyltransferase (B). Lane T1, ladder digested by RNase T1 under denaturing conditions; lane OH − , alkaline digestion. (C) Structure analysis by enzymatic probing. tRNA regions where significant reactivity changes occurred are highlighted in grey. sugar-phosphate backbone of the tRNA [38,39]. In addition, significant expansion and contraction of the RNA helix have been observed with significant changes of the helical conformation of the T-stem at bps 50/64 and 51/63 in the immediate vicinity of the pathogenic G52A mutation that disrupts the GC Watson Crick bp [40]. Mutations in hmtRNALeu (CUN) interfere with m1 G37 modification TrmD and Trm5 are the bacterial and eukarya/archaea methyl transferases, respectively, that catalyse the transfer of the methyl group from AdoMet to the N1 position of G37 in tRNA to synthesize m1 G37 -tRNA. The m1 G37 modification prevents tRNA frameshifts on the ribosome by assuring the correct codon– anticodon pairings and thus is essential for the fidelity of protein synthesis. In addition, m1 G37 contributes to efficient and accurate aminoacylation [41–43]. Trm5 interacts directly with the outer corner in the L-shape of the tRNA to check if the tertiary structure of the tRNA is adequate (Figure 6A) [44], TrmD can methylate a stem-loop RNA with 9 bp c The Authors Journal compilation c 2013 Biochemical Society and, therefore, has no requirement for the L-shape of tRNA in the catalysis [30,31]. In the present study, we used this mechanism of recognition of the outer corner to probe the tertiary-structure of the mutated transcripts of tRNALeu (CUN) harbouring G37 . Methylation of the transcripts was assayed and compared in the presence of Trm5 and TrmD. With Trm5, the G52A mutant of tRNALeu (CUN) induced significant decreases in the methylation properties with a plateau value reaching only 30 % that of the wild-type transcript, whereas the A57G mutant was methylated to levels comparable with the wild-type transcript (Figure 6B). This result confirmed that the G52A mutation induced conformational changes in the L-shape of tRNA, as shown above by probing. When performing the methylation assay in the presence of E. coli TrmD, the homologue of Trm5 that does not recognize the L-shape of RNA but just the anticodon stem [31,45], the three tRNALeu (CUN) transcripts were methylated with comparable efficiencies (Figure 6C). This result suggested that the two mutations did not significantly impact the structure of the anticodon loop of the two mutated transcripts. Functional and structural defects in tRNA mutants Figure 5 461 CCA addition by hmTNT and impact of the G52A and A57G mutations on the reaction (A) CCA addition was monitored by the incorporation of [32 P]ATP at position 76 of tRNA. Reaction mixtures were fractionated by denaturing PAGE and radioactivity was detected and quantified by imaging. (B) Time curves of CCA adding reactions indicated that G52A mutants were very weak substrates for hmTNT, whereas the A57G mutants were nearly as active as the wild-type tRNAs (WT). (C) Crystal structures of Archaeoglobus fulgidus tRNA nucleotidyltransferases with tRNA (PDB code 1SZ1). The enzyme is represented as a cartoon model in grey. The tRNA is shown in dark in the cartoon model with the G52 and A57 nucleotides in white. Results are reported as averages of three assays and error bars indicate S.D. Table 1 Kinetic constants of hmTNT in the CCA-adding reaction at 37 ◦ C All rates represent the average of three assays + − S.D. Isoacceptor Mutation K m (μM) k cat (min − 1 ) K cat /K m (min − 1 ·μM − 1 ) N-fold reduction hmtRNALeu (CUN) Wild-type G52A A57G Wild-type G52A A57G 1.95 + − 0.05 2.33 + − 0.15 3.10 + − 0.42 1.11 + − 0.08 9.64 + − 0.05 2.09 + − 0.15 −2 (9.13 + − 0.98)× 10 − 3 (8.33 + − 0.06)× 10 − 2 (7.43 + − 0.34)× 10 − 1 (3.21 + − 0.08)× 10 − 1 (2.60 + − 0.10)× 10 − 1 (4.58 + − 0.25)× 10 0.044 0.0037 0.024 0.29 0.027 0.22 1 11.9* 1.8* 1 10.7† 1.3† hmtRNALeu (UUR) *Loss of catalytic efficiency relative to wild-type hmtRNALeu (CUN). †Loss of catalytic efficiency relative to wild-type hmtRNALeu (UUR). G52A mutation in hmtRNALeu (UUR) interferes with i6 A37 modification MiaA catalyses the addition of an isopentenyl group from DMAPP to the N6 -nitrogen of A37 . Almost all tRNAs that read codons starting with U have the modified nucleotide i6 A37 in the anticodon loop, including tRNAPhe , tRNATrp , tRNATyr , tRNACys , tRNALeu and tRNASer , that are further methylthiolated by the action of the MiaB [(dimethylallyl) adenosine tRNA methylthiotransferase] and MiaC enzyme activities to form ms2 i6 A37 . The ms2 i6 A37 modification is thought to stabilize tRNA–mRNA interactions by improving intrastrand stacking within tRNA anticodon loops and interstrand stacking between codons and anticodons [46]. During enzyme catalysis, MiaA binds the anticodon helix–loop composed of nucleotides A26 C41 and specifically recognises the canonical triple A36 A37 A38 found in the tRNAs to be modified (Figure 6D) [47]. In the present study, we performed modification assays on hmtRNAsLeu (UUR) using purified E. coli MiaA enzyme and DMAPP as a substrate. The wild-type transcript and A57G mutant were equally modified; however, the G52A transcript was poorly modified with a plateau value reaching about 30 % that of the wild-type transcript (Figure 6E). This result suggested that the MiaA enzyme was sensitive to the G52A mutation, despite the fact that the mutation was located at a distance of more than 50 Å from A37 that is modified. In fact, the MiaA enzyme tightly clamps the anticodon helix between its catalytic domain and swinging domain. During the binding, the anticodon proximal stem is critical because, from the 29/41 and 31/39 bps, the rift formed between the two protein domains is very narrow, allowing only tight base pairing in the anticodon proximal stem [47,48]. c The Authors Journal compilation c 2013 Biochemical Society 462 M. Wang and others Figure 6 Methylation and isopentenylation of wild-type and mutated hmtRNAsLeu by eukaryal m1 G-methyl transferase Trm5, bacterial TrmD and bacterial isopentenyl-tRNA transferase MiaA (A) Crystal structures of Methanocaldococcus jannaschii methyl transferases with tRNACys (PDB code 2ZZN). The enzyme is represented as a cartoon model in grey. The tRNA is shown in dark in the cartoon model with G52 and A57 nucleotides in white. Time curve of methylation of tRNAsLeu (CUN) by human Trm5 (B) and E. coli TrmD (C). (D) Crystal structures of E. coli tRNA prenyltransferase with tRNA (PDB code 3FOZ). The enzyme is represented as a cartoon model in grey. The tRNA is shown in black in the cartoon model with the G52 and A57 nucleotides in white. (E) Time curve of incorporation of isopentenyl moiety in tRNAsLeu (UUR) by E. coli MiaA. Results are reported as averages of three assays and error bars indicate S.D. hmEF-Tu protection is reduced for the four hmtRNALeu s mutants DISCUSSION EF-Tu binds all elongator aa-tRNAs for delivery to the ribosome during protein synthesis. Functioning as an aa-tRNA synthetase, EF-Tu contributes to translation accuracy by binding tRNAs using a thermodynamic compensation mechanism, which allows verification that the tRNAs are correctly acylated by their corresponding amino acids [49]. Once bound to EF-Tu, the ester bond of aa-tRNAs is protected against spontaneous hydrolysis and protection is maintained until incorporation of the amino acid moiety into the nascent peptide chain. On EF-Tu, tRNA is bound by its 3 - and 5 -ends, and the T-stem interacts with a distinct domain of the factor (Figure 7A) [50]. Therefore hmEFTu binding is sensitive to T-stem structure defects as shown by pathogenic mitochondrial mutations [51]. In the present study, we monitored hmEF-Tu protection for the various transcripts and compared it with spontaneous hydrolysis without the factor. In the scaffolds of both tRNALeu (CUN) and tRNALeu (UUR), the A57G mutation induced rapid deacylation identical to that measured in the absence of hmEF-Tu. This finding suggested that the A57G mutation modified the T-stem structure in a way that rendered it incompatible with hmEF-Tu binding (Figures 7B and 7C). The G52A mutation induced in the two tRNA scaffolds an intermediate level of protection between those of the wild-type tRNA and A57G mutants. hmtRNAsLeu , especially tRNALeu (UUR) which also contains a transcription termination sequence, is a hot spot for point mutations associated with variable clinical phenotypes as diverse as MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), cardiomyopathy, CPEO, diabetes, deafness and more complex syndromes. In the present study, by using aminoacylation to probe the structure–function properties of the pathogenic mutations, we selected four mutations that drastically decreased the charging capacity. Mutations G52A in the T-stem and A57G in the T-loop showed comparable aminoacylation deficiencies in both tRNALeu (CUN) and tRNALeu (UUR). The G52A mutation introduced an A52 C62 pair in the Tstem instead of a strong G52 C62 pair, suggesting that the T-arm structure could be weakened. mtRNAs contain an unusually high percentage of AU bases which inherently decrease the tRNA stability. Several examples of ‘slippage’ of bases in the secondary structures of mitochondrial tRNAs [24,52], even those with considerable conformational rearrangements [53,54], have been reported. In the present study, the pathogenic mutations of G52A in the two tRNAsLeu isoacceptors may have induced a comparable T-stem fragility, as shown by new accessibility to RNases T1, V1 and S1 in the outer corner of tRNA. This more distant effect may be attributed to the conformation of the T-arm modifying the c The Authors Journal compilation c 2013 Biochemical Society Functional and structural defects in tRNA mutants Figure 7 463 Protection of hmtRNAsLeu against deacylation by hmEF-Tu (A) Crystal structure of E. coli EF-Tu with tRNAPhe (PDB code 1OB2). The enzyme is represented as a cartoon model in grey. The tRNA is shown in black in the cartoon model with the G52 and A57 nucleotides in white. (B) Preformed [14 C]Leu–tRNAsLeu (103 c.p.m., 1 μM) was incubated with the purified hmEF-Tu–GTP complex. Protection against deacylation was measured after various incubation times at 37 ◦ C. (C) Similar reactions were performed in the absence of hmEF-Tu in order to monitor the spontaneous deacylation. Results are reported as averages of three assays and error bars indicate S.D. WT, wild-type. tertiary core of the tRNA via long-range interactions between the D, T and V-loops, resulting in a conformational change of the anticodon arm which stacks co-axially to the D-arm domain. Besides causing a dramatic decrease in aminoacylation efficiency, the G52A mutation also altered the 3 -end processing by tRNA nucleotidyl transferase. The substitution also precluded the modification of nucleotide 37 by Trm5 and MiaA, as well as the binding to hmEF-Tu. Most of these reactions are based on protein interactions with the outer corner of the tRNA, which seems to be critically modified in the two mutated isoacceptors. The A57G mutation only altered an unpaired base in the Tloop of the two tRNA isoacceptors. However, it induced drastic decreases of the aminoacylation comparable with that with the G52A mutation. Analyses of the melting curve of tRNALeu and native gels highlighted subtle differences in the structure that could not be revealed by enzymatic probing, but could affect the interaction with the aa-tRNA synthetase. Similar decreases in the aminoacylation efficiency were reported for pathogenic mutations found in the T-loop of hmtRNAs. U60C in tRNALeu (UUR) was shown to reduce the aminoacylation efficiency to a nearly undetectable level, and it could be a competitive inhibitor for the homologous wild-type tRNA [11]; A59G in tRNAIle exhibited a very low activity, and it was proposed that the substitution might perturb the secondary structure by shifting the base pairing so that the T-loop becomes reduced in size by three nucleotides [52]. Similarly, hmEF-Tu, which binds in the L structure at the top helix formed by the acceptor stem co-axially stacked to the T-stem-loop domain, is also sensitive to the A57G mutation. Usually, in cytosolic tRNAs, base 57 is located in a region rich of tertiary pairs G18 55 , G19 C56 and U54 A58 (Figure 1B), and therefore it contributes to tRNA core formation. As the C-terminal domain of LeuRS binds the outer corner of the tRNA [55], it may be sensitive to the conformational changes, and the binding capacity could be decreased with the A57G mutants. Given the central role of the aminoacylation of tRNAs in protein synthesis, these physiological phenotypes may induce perturbations in the efficiency of translation. It was shown that the substantial decrease in aa-tRNA is connected to the decrease in mitochondrial protein synthesis due to premature termination of translation [56,57]. Mitochondria are heteroplasmic, and populations of both mutant and wild-type tRNAs co-exist within a single mitochondrium. The impairment of mitochondrial protein translation is only apparent when the percentage of mutated tRNA exceeds a threshold value, which takes into account many parameters. The wild-type tRNA may rescue the synthesis, and the mutated tRNA may be a competitive inhibitor for substrates or other interacting partners. Altogether, the collected data show that G52A and A57G mutations in the T-arm domain induced multiple effects on tRNA metabolism. They influenced not only aminoacylation, but also the ability of the tRNA to be a substrate for processing or modification enzymes or to function in subsequent steps of protein synthesis. Faced with a situation with wide impacts at different levels of the translation process, a treatment strategy for mitochondrial diseases to be considered would be to target the cause of the disorders, such as the import of mitochondrially targeted functional aa-tRNA synthetase [18] or tRNAs [19]. AUTHOR CONTRIBUTION En-Duo Wang and Gilbert Eriani designed the research. Meng Wang performed the research. Meng Wang, Xiao-Long Zhou, Ru-Juan Liu, Zhi-Peng Fang, Mi Zhou, Gilbert Eriani and En-Duo Wang analysed the data. Meng Wang, Gilbert Eriani and En-Duo Wang wrote the paper. ACKNOWLEDGEMENTS We thank Dr Eric Ennifar and Dr Philippe Dumas for advice in tRNA melting experiments and interpretations. We thank Dr Franck Martin for constant advice and Professor Catherine Florentz for helpful discussion and encouragement. FUNDING This work was supported by the National Key Basic Research Foundation of China [grant number 2012CB911000], the Natural Science Foundation of China [grant numbers c The Authors Journal compilation c 2013 Biochemical Society 464 M. 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(2013) 453, 455–465 (Printed in Great Britain) doi:10.1042/BJ20130294 SUPPLEMENTARY ONLINE DATA Multilevel functional and structural defects induced by two pathogenic mitochondrial tRNA mutations Meng WANG*†, Xiao-Long ZHOU*, Ru-Juan LIU*, Zhi-Peng FANG*, Mi ZHOU*, Gilbert ERIANI†1 and En-Duo WANG*1 *Center for RNA Research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China, and †Architecture et Réactivité de l’ARN, Université de Strasbourg, Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France Figure S1 Measurement of equilibrium dissociation constants for tRNA by filter-binding assays The P-labelled hmtRNALeu (6500 c.p.m., ∼ 2.5 pmoles) was incubated in a 50 μl reaction volume in the presence of hmLeuRS at various concentrations for 15–30 min at 0 ◦ C in a buffer containing 50 mM Hepes/KOH (pH 6.8), 30 mM KCl and 12 mM MgCl2 . Then the samples were applied and filtered through a nitrocellulose membrane. The filters were washed with 0.3 ml of washing buffer, dried and the radioactivity was counted. Data were analysed using GraphPad PRISM software. WT, wild-type. 32 Figure S2 UV melting curves of the two tRNAsLeu isoacceptors and mutated derivatives tRNAs (∼ 0.7 μM final concentration) were diluted in 50 mM potassium phosphate buffer (pH 7.0) with 100 mM NaCl and 0.1 mM EDTA. Absorbance against temperature melting curves were measured at 260 nm with a heating rate of 0.2 ◦ C/min from 20 to 90 ◦ C on a UVIKON-XL spectrometer equipped with a Peltier thermostated cell holder. Melting experiments were repeated three times. After reaching 90 ◦ C, the temperature was progressively cooled to 20 ◦ C at a rate of 0.2 ◦ C/min. Absorbance was monitored as before at 260 nm. WT, wild-type. Received 26 February 2013/24 April 2013; accepted 1 May 2013 Published as BJ Immediate Publication 1 May 2013, doi:10.1042/BJ20130294 1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]). c The Authors Journal compilation c 2013 Biochemical Society