Download Multilevel functional and structural defects induced by two

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. Wang and others
30930022, 31000355 and 31130064], the Committee of Science and Technology in
Shanghai [grant number 12JC1409700], the Centre National de la Recherche Scientific
(CNRS), a Visiting Professorship for Senior International Scientists from the Chinese
Academy of Sciences [grant number 2011T2S10] and the Joint Doctoral Promotion
Program of the Chinese Academy of Sciences (Sino-France) (to W.M.).
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Received 26 February 2013/24 April 2013; accepted 1 May 2013
Published as BJ Immediate Publication 1 May 2013, doi:10.1042/BJ20130294
c The Authors Journal compilation c 2013 Biochemical Society
Biochem. J. (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