Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
Nucleic Acids Research
Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells
Marina Toompuu 1
Tea Tuomela 1
Pia Laine 0
Lars Paulin 0
Eric Dufour 1
T. Jacobs 0 1
0 Institute of Biotechnology, FI-00014 University of Helsinki , Finland
1 Faculty of Medicine and Life Sciences, BioMediTech Institute and Tampere University Hospital, FI-33014 University of Tampere , Finland
RNA 3 polyadenylation is known to serve diverse purposes in biology, in particular, regulating mRNA stability and translation. Here we determined that, upon exposure to high levels of the intercalating agent ethidium bromide (EtBr), greater than those required to suppress mitochondrial transcription, mitochondrial tRNAs in human cells became polyadenylated. Relaxation of the inducing stress led to rapid turnover of the polyadenylated tRNAs. The extent, kinetics and duration of tRNA polyadenylation were EtBr dose-dependent, with mitochondrial tRNAs differentially sensitive to the stress. RNA interference and inhibitor studies indicated that ongoing mitochondrial ATP synthesis, plus the mitochondrial poly(A) polymerase and SUV3 helicase were required for tRNA polyadenylation, while polynucleotide phosphorylase counteracted the process and was needed, along with SUV3, for degradation of the polyadenylated tRNAs. Doxycycline treatment inhibited both tRNA polyadenylation and turnover, suggesting a possible involvement of the mitoribosome, although other translational inhibitors had only minor effects. The dysfunctional tRNALeu(UUR) bearing the pathological A3243G mutation was constitutively polyadenylated at a low level, but this was markedly enhanced after doxycycline treatment. We propose that polyadenylation of structurally and functionally abnormal mitochondrial tRNAs entrains their PNPase/SUV3-mediated destruction, and that this pathway could play an important role in mitochondrial diseases associated with tRNA mutations.
RNA 3 polyadenylation has been reported to serve many
roles in biology, with a distinction usually drawn between
eukaryotes, where poly(A) is considered to play a positive
role, facilitating nuclear export, stability and translation,
and prokaryotes, where poly(A) is typically used as a tag
to mark RNAs for degradation (reviewed in (
eukaryotic organelles, notably chloroplasts, the bacterial
principle of poly(A)-dependent RNA degradation prevails,
although poly(A) plays a more ambiguous and often
taxonor even gene-specific role in mitochondria (
metazoan mitochondria, polyadenylation has been variously
inferred to promote either mRNA turnover or stabilization
), translation (
), tRNA maturation and/or repair
) and to play a role, most likely an indirect one, in the
sensitivity of nuclear DNA to double-strand breaks induced
by ionizing radiation (13).
The dichotomous effects of 3 poly(A) on metazoan
mitochondrial mRNAs (stabilization versus destabilization)
appear to be transcript-specific, with some stabilized but
others destabilized by the inhibition of polyadenylation
). The addition of A residues is also formally
necessary for the creation of some UAA stop codons (16).
The enzyme responsible for the synthesis of the poly(A)
tails of mitochondrial mRNAs is the mitochondrial poly(A)
polymerase (mtPAP, product of the PAPD1 gene). mtPAP
may also be involved in the oligouridylation of histone
mRNAs in the cytoplasm at the termination of S-phase
). The degradation of human mitochondrial RNAs
tagged with poly(A) is catalyzed by the components of
the mitochondrial ‘degradosome’, namely SUV3 helicase
(SUV3L1 gene product) and polynucleotide phosphorylase
(PNPase, PNPT1 gene product, (
)). Degradation is
believed to initiate when this complex interacts with unfolded
poly(A) tails: thus, in the absence of SUV3 function,
abnormal polyadenylated RNAs accumulate (
Oligoadenylated tRNAs are detected when the processing or
surveillance enzyme PDE12 is knocked down (
). Low level
adenylation of misprocessed or truncated RNAs, including
tRNAs, has been reported even in control cells (7).
Polyadenylation of tRNAs has been documented in
bacteria, under the abnormal conditions of deficiency of tRNA
processing enzymes (
) or deregulation of the poly(A)
polymerase PAP I (23), leading to tRNA destruction and
severely impaired protein synthesis. Defective tRNAs are
tagged for degradation by polyadenylation in Escherichia
) and eukaryotic nuclei (
). Polyadenylated tRNAs
have also been reported in chloroplasts (
), although their
physiological meaning is unknown.
In this study, we investigated the effects of ethidium
bromide (EtBr), a DNA-intercalating agent that suppresses
mitochondrial transcription. EtBr also intercalates into RNA,
including tRNA (
), but this intercalation is restricted by
the adoption of tertiary structures. In high-salt conditions
tRNAs retain their native L-form structure, and are
typically able to bind about tenfold less EtBr than when the
tertiary structure is disturbed (
), with a preferred binding
site at the base of the acceptor stem (
In previous studies, we (
) and others (
) have used
EtBr at doses of up to 250 ng/ml to suppress mitochondrial
transcription, resulting in the rapid disappearance of
mitochondrial mRNAs and the gradual decay of the more
stable mitochondrial rRNAs and tRNAs. Noting that higher
doses of EtBr should intercalate more effectively into
tRNAs, thus distorting their structure, we investigated the
effects on mitochondrial tRNA metabolism of subjecting
cells to tenfold greater concentrations of the drug than those
used previously. Under these conditions, we revealed an
unexpected propensity of mitochondrial tRNAs to acquire
long poly(A) tails. After withdrawal of the EtBr,
polyadenylated tRNAs were rapidly degraded, following a lag phase.
We propose that this represents a mitochondrial
surveillance system for abnormal tRNAs, similar to the tRNA
quality-control process previously documented in bacteria.
MATERIALS AND METHODS
Cell lines and culture
Previously described cell lines were as follows: 143B
osteosarcoma cybrid cell lines homoplasmic for wild-type
mitochondrial DNA (mtDNA; clone 43) or the 7472insC
mutation (clone 47) (
), A549 lung carcinoma cells and the
GT cybrid cell line derived from the A549 background,
containing both np 3243 and np 12300 mutant mtDNA (35).
Cells were routinely cultured in DMEM with or without
uridine and pyruvate as described previously (
Oligonucleotides and reagents
Custom-designed DNA oligonucleotides were purchased
from DNA Technology (Aarhus, Denmark). Their
sequences and other relevant information are shown in
Supplementary Table S1. Inhibitors used in polyadenylation
assays at the final concentrations shown in figures and
legends were cordycepin (3 deoxyadenosine), CCCP,
doxycycline, oligomycin (mixture A, B, C), or thiamphenicol (all
from Sigma), or puromycin (InvivoGen). Antibodies were
from Abcam: anti-mtPAP (ab154555, used at 1:2000);
antiPDE12 (ab87738, 1:500); anti-PNPT1 (PNPase, ab96176,
1:2000) and anti-SUV3L1 (SUV3, ab176854, 1:2000).
AntiGAPDH antibody was from Cell Signaling Technology
(14C10, #2118, 1:3000), anti- -actin from Santa Cruz
(C4, sc-47778, 1:200) and HRP-conjugated goat
antirabbit (111-035-144) and goat anti-mouse (115-035-146)
secondary antibodies (1:20 000) were from Jackson
ImmunoResearch Inc., the latter being used for detection of
the anti-actin signal.
Total RNA was extracted from cells using the Trizol reagent
(Life Technologies) under manufacturer’s recommended
conditions. RNA was fractionated on 12% polyacrylamide
(PAGE)-7 M urea/TBE gels, electroblotted to Zeta-Probe
or Zeta-probe GT membranes (BioRad), and hybridized to
gene-specific oligoprobes as described previously (
using Rapid-hyb buffer (GE Healthcare). Signals were
visualized by sensitive X-ray film or, where indicated in
legends, by phosphorimaging (Typhoon™ imaging system, GE
Healthcare). For aminoacylation analyses, total RNA was
dissolved on ice in 0.1 M sodium acetate, pH 5.2 and
fractionated on acidic 6.5% PAGE-7 M urea gels essentially as
described previously (
). Samples were deacylated by
heating for 10 min at 75◦C followed by 30 min at 37◦C in
1.5 volumes of 0.5 M Tris–HCl, pH 9.0. To analyze the
primary structure of tRNA products in EtBr-treated cells,
total RNA was isolated, tRNAs deacylated and
circularized by T4 RNA ligase (MBI Fermentas) as described (
reverse-transcribed with oligonucleotides cser1 or cleu1 and
PCR-amplified using oligonucleotides cser1 and cser2 for
analysis of tRNASer(UCN) or cleu1 and cleu2 for analysis of
tRNALeu(UUR), essentially as described previously (
high-molecular weight tRNALeu(UUR) products induced by
doxycycline treatment were gel-extracted, circularized and
amplified similarly. PCR products were cloned (TOPO TA
cloning kit, Invitrogen) and inserts Sanger-sequenced using
M13 forward primer and standard dye-terminator
technology as described previously (
Analysis of tRNA response to EtBr
Cells were seeded at equal densities on 6 cm plates, giving
70–80% confluence, 14–16 h before the experiments. They
were then incubated in fresh medium containing different
concentrations of EtBr for the times indicated in figures and
legends. Inhibitors of the respiratory chain or translation
were added to fresh medium (concentration indicated in
figures and legends) 1 h before treatment with EtBr. CCCP
treatment was also performed in the reverse order,
following 1 h of pre-incubation of cells with 5 g/ml EtBr. For
siRNA depletion experiments, cells were seeded on 10 cm
plates and transfected with 100 nM siRNAs and
Lipofectamine RNAiMAX (Invitrogen) under manufacturer’s
recommended conditions. Two days after transfection (or on
day 3, see legends), cells were seeded at equal densities on 6
cm plates and EtBr was added one day later.
Verification of siRNA-mediated knockdown by Western blotting
The efficiency of mRNA depletion by siRNA-based RNA
interference (see Supplementary Table S1 for siRNA
sequences) was analyzed at the protein level by Western
blotting. Cells were lysed in PBS containing 1% N-dodecyl-
D-maltoside (or 1% SDS where indicated), 1 mM PMSF
and Pierce protease inhibitor (Thermo Scientific),
incubated on ice for 30 min and centrifuged for 20 min at
20 000 gmax (
). Protein concentrations were determined
by the DC protein assay (BioRad), fractionated on 10%
SDS-PAGE (30 g protein per lane) and electroblotted
to Whatman® Protran® nitrocellulose membrane (Perkin
Elmer). Membranes were blocked in 5% non-fat dried
milk/0.1% Tween/TBS followed by incubation with
primary and HRP-conjugated secondary antibodies at the
dilutions indicated above. Signals were analyzed using the
ChemiDoc imaging system (BioRad).
Cell growth and viability
Cultures of 106 cells were seeded in 10 cm cell plates. After
24 h, 2.5 g/ml of EtBr was added and cells were grown for
a further 24 h, then harvested and counted using EVE™
automated cell counter (NanoEnTek), with cell viability
determined by trypan-blue exclusion.
Plates of 3 × 106 cells were treated for 24 h with 2.5 g/ml
EtBr. Intact cell respiration in fresh, EtBr-free medium at
37◦C, and in permeabilized cells supplied with cI-
cIIIor cIV-linked substrate mixtures, was determined using a
Hansatech oxygraph as described previously (
), with all
reagents sourced from Sigma-Aldrich. To assess coupling of
respiration, permeabilized cells supplied with the cI-linked
substrate mixture were treated successively with ADP,
followed by oligomycin at concentrations ranging from 25 to
75 nM, and FCCP at concentrations ranging from 0.5 to 2
M, in both cases selecting for further analysis the
respective concentration leading to maximal inhibition or
activation of oxygen consumption.
mtDNA copy number analysis
Quantification of total mtDNA was performed essentially
as described (
), by comparing the efficiencies of
amplification of mitochondrial ND1 or ND4 (
) with the
nuclear multi-copy gene encoding 18S rRNA (
was extracted with QIAmp DNA mini kit (QIAgen) from
cultured cells according to manufacturer’s instructions. 10
ng of DNA was used per qPCR reaction, using primers (300
nM) and fluorogenic probes (50 nM), both from Metabion
International, as listed in Supplementary Table S1, and
TaqMan® Universal PCR Master Mix (Applied
Biosystems). Each reaction was performed in triplicate and in two
independent runs, using the following profile: one cycle at
95◦C for 20 s and then 40 cycles at 95◦C for 3 s and 60◦C
for 30 s. Threshold cycle numbers (Ct) were calculated with
StepOnePlus v3.1 program (Applied Biosystems) and the
results from the two runs were averaged. The ND4/18S and
ND1/18S ratios were calculated from cycle threshold
values, and normalized to the values from untreated cells.
diluted to 1.5 ml. After a further 24 h, cells were washed,
then stained in 0.5 ml of 2 M CM-H2DCF-DA
(ThermoFisher Scientific) in PBS for 60 min at 37◦C in the dark.
After washing with 1 ml warmed PBS, cells were detached in
80 l trypsin–EDTA solution (0.25%, Sigma-Aldrich), for 3
min at 37◦C, after which 0.5 ml of fresh medium was added
and cells, maintained in the dark throughout, were analyzed
with an Accuri® C6 flow cytometer (BD Biosciences)
using 488 laser excitation, fast flow and limits set at 30 000
live cells (ROI selection based on forward & side scatter)
or 2 min. Corrections for channel crosstalk were estimated
from single dye measurements: DCF signal = FL1(533/30)
– 2.8% of FL3; EtBr signal = FL3(670/LP) – 1.1% of FL1.
Analysis of mitochondrial translation products
Mitochondrial translation products were pulse-labeled with
35S-methionine in the presence of emetine, and analyzed as
described previously (
) by SDS-14% PAGE.
Library preparation for targeted next-generation sequencing
Illumina TruSeq truncated reverse primer 5 Phos-NNA
C-Amino 3 was first adenylated using a 5 DNA
Adenylation Kit (New England Biolabs). The adenylated oligo (5
pmol) was ligated to total RNA (500 ng) using truncated
T4 RNA Ligase 2 (200 U, New England Biolabs) in a 10 l
reaction. cDNA was generated using a primer
complementary to the ligated reverse primer, with Superscipt II RNA
reverse transcriptase (400 U, ThermoFisher Scientific)
in a 22 l reaction. Primers were designed for human
mitochondrial tRNAs (GenBank entry NC 012920) and
the truncated Illumina TruSeq forward adapter sequence
3 was added to the 5 end of each of the specific primers
(see Supplementary Table S1). Amplification of the selected
cDNAs was performed using a 2-step PCR approach. In
the first step the designed specific primers with overhangs
and Illumina Index primers, 10 pmol each, were used
for amplification, with 5 l of cDNA as template, using
Phusion HotStart II DNA Polymerase (ThermoFisher
Scientific) with an activation step of 98◦C for 10 s and 18
cycles of 98◦C for 10 s, 55◦C for 30 s and 72◦C for 10 s. The
obtained PCR products were then treated with Exonuclease
I and FastaAP Phosphatase (both from ThermoFisher
Scientific) to remove unused primers and nucleotides. An
aliquot of the first PCR (5 l) was used as template for
the second PCR in order to incorporate the full-length
Illumina adapters for the final library, using the same PCR
cycle as above. The obtained products were pooled and
purified using AMPure XP (Beckman Coulter). Size selection
was done using BluePippin (Sage Sciences) with a cut-off
of 220–550 bp. The final pool was checked on Fragment
Analyzer (Advanced Analytical) and concentrations were
measured using Qubit (Life Technologies).
Measurement of whole-cell ROS levels by DCF fluorescence
DNA sequence analysis
Cells were seeded in 12-well plates at 105 cells per well (1
ml). After 24 h, 2.5 g/ml EtBr was added and medium
The Illumina MiSeq System was used to sequence
samples in paired end (R1 and R2) manner. Obtained reads
of 326 bp (R1) and 286 bp (R2) were trimmed using
cutadapt (v1.7.1; 46) with default parameters, except that
minimum read length (m) was set to 50 bp and the quality
minimum (q) to 20. In addition, Illumina’s Truseq adapter
sequences were removed. Reads were trimmed in paired-end
manner to keep both read pairs following the criteria
defined above. Trimmed paired-end reads were overlapped
using FLASH (fast length adjustment of short reads) tool
). The BLASTN tool (
) was used to divide sequences
into different tRNA sequence pools. Within each tRNA
sequence pool, sequences were sorted and unique sequences
were counted, applying exclusion criteria as indicated in
Blot images were optimized for brightness and contrast and
cropped, rotated and/or framed for clarity, but no other
manipulations were applied.
High doses of EtBr induce polyadenylation of human mitochondrial tRNAs
Treatment with 0.25 g/ml EtBr is sufficient to inhibit
mitochondrial RNA synthesis in 143B osteosarcoma cell
cybrids, as we showed previously (
). In preliminary
experiments, we observed that treatment of cells with a 10-fold
higher dose of the drug resulted in altered mobility of
mitochondrial tRNAs on denaturing polyacrylamide gels. We
investigated this phenomenon in the same 143B cybrids as
in our earlier study, which were homoplasmic, respectively,
for wild-type mtDNA and for 7472insC mutant mtDNA
). Exposure of cells to a concentration of 2.5 g/ml EtBr
for 8 h or more resulted in a shift of most of the pool
of tRNASer(UCN) to species of higher apparent molecular
weight (Figure 1A). The process had already started after
4 h of treatment. By 24 h hardly any of the original tRNA
band remained on Northern blots, but much of the modified
species had also been degraded (Figure 1A–C). The process
had similar kinetics in wild-type and mutant cybrids
(Figure 1B, C, Supplementary Figure S1A), despite the lower
steady-state abundance of the mutant tRNA ((
In contrast, cytosolic tRNASer(UCH) was unaffected by
EtBr treatment (Figure 1A). The electrophoretic mobility
of a typical mitochondrial mRNA, such as for ND3, was
also unmodified, but it was progressively degraded
(Supplementary Figure S1B), as expected given the known effects
of EtBr on mitochondrial transcription and the relatively
short half-lives of mitochondrial mRNAs.
The effects of such high levels of EtBr on
mitochondrial functions and cell physiology have not previously
been tested. We therefore confirmed that treatment with 2.5
g/ml EtBr over 24 h did not impair cell viability
(Supplementary Figure S1C), although cell proliferation was
curtailed (Supplementary Figure S1C). Under these
conditions, the copy number of mtDNA (Supplementary
Figure S1D) and the rate of respiration of both intact and
permeabilized cells (Supplementary Figure S1E) declined
by ∼50%. Although measurements of membrane potential
and mitochondrial ROS production by standard
fluorimetric methods were not possible, due to the interference of
EtBr fluorescence with that of TMRM or MitoSox, we were
able to assess these parameters indirectly. The respiration
of permeabilized EtBr-treated cells was stimulated by ADP
or by the uncoupler FCCP, and the proportionate
inhibition by oligomycin was the same as in control cells
(Supplementary Figure S1F), indicating that the treatment did
not result in uncoupling or major changes in membrane
potential. Estimates of whole-cell ROS based on DCF
fluorescence, which were not subject to interference from EtBr
(Supplementary Figure S1G), indicated that EtBr treatment
did result in substantially increased ROS (Supplementary
Figure S1G). Finally, in accord with its hypothesized effects
on RNA, EtBr treatment abrogated mitochondrial protein
synthesis even more effectively than doxycycline
(Supplementary Figure S1H).
Next, we tested other mitochondrial tRNAs (Figure 2,
Supplementary Figure S2A–E). These appeared to vary
in their susceptibility to EtBr-induced modification and
turnover, and the kinetics thereof. Of those tested, tRNAPhe
was the most sensitive to EtBr treatment, with the mature
tRNA having almost disappeared after 8 h (Figure 2B,
Supplementary Figure S2A), but most of it already modified
by 4 h of treatment. At the other end of the spectrum was
tRNASer(AGY), approximately 10% of which remained as
the mature tRNA even after 24 h, and where the relative
amount of the modified tRNA never reached 50% or above.
Of the other mitochondrial tRNAs tested, each appeared
to respond to treatment with high levels of EtBr with
specific characteristics. tRNAGln showed a similar
susceptibility to EtBr-induced modification as tRNASer(UCN), while
tRNAHis behaved like tRNASer(AGY), and tRNALys and
tRNATrp were intermediate (Supplementary Figure S2B).
Mature tRNALeu(UUR) turned over slowly like tRNASer(AGY)
but a larger fraction became modified (Figure 2B,
Supplementary Figure S2A).
The set represents both clustered and individual
tRNAs, tRNAs encoded on each strand of mtDNA, frequent
pathological targets, and both the most ‘canonical’ and the
most structurally aberrant human mitochondrial tRNAs
(tRNALeu(UUR) and tRNASer(AGY), respectively). None of
these features correlated in a straightforward manner with
susceptibility to EtBr. The extent of modification was EtBr
dose-dependent, with 5 or 10 g/ml EtBr giving a more
pronounced response than 2.5 g/ml (e.g. Figure 3A, C,
Supplementary Figure S2D). Apart from tRNASer(UCN), which
differed only in abundance, other tRNAs behaved similarly
in wild-type and 7472insC mutant cybrids (Supplementary
Figure S2C). A similar response was observed in A549 lung
carcinoma cells, following treatment with 5 g/ml EtBr
(Supplementary Figure S2E).
To identify the nature of the novel tRNA products in
EtBr-treated cells, total RNA from wild-type cybrid cells
was isolated, tRNAs were deacylated, and sequences
analyzed initially by Sanger sequencing of cloned,
circularized RNAs amplified with tRNA-specific oligonucleotides.
For each of tRNASer(UCN) and tRNALeu(UUR) we obtained
sequences from >20 clones of 3 -tailed molecules. The
extensions consisted almost exclusively of poly(A), with tails
of up to 61 nt for tRNASer(UCN) and up to 38 nt for
tRNALeu(UUR), some of which were truncated at or within
the 3 -terminal CCA. Polyadenylated tRNAs were detected
only in EtBr-treated cells: for comparable analyses from
untreated control cells see (
To obtain a more reliable estimate of the composition
of the tails, we employed targeted next-generation
sequencing (tNGS) for these and several other mitochondrial
tRNAs (Table 1), generating thousands of independent
sequence reads (Supplementary Table S2). Non-A residues
comprised less than 1% of the total for each of the
analyzed tRNAs, but with some variability between them,
with tRNASer(UCN) showing the lowest proportion amongst
those analyzed, as well as the lowest proportion of
truncation at or within the CCA. tNGS revealed 3 tails of up to 51
nt (Table 1, Supplementary Table S2), although this is likely
an underestimate, due to the inherent bias of the method
towards shorter tailed molecules. Circularization and cloning
introduce a similar bias.
Based on the electrophoretic mobility of RNA size
markers, tailed tRNASer(UCN) species ranged in apparent
strandlength from the size of the mature tRNA up to
approximately 150 nt, after 6 h of treatment with 2.5 g/ml EtBr.
Their size increased further by 11 h of such treatment
(Supplementary Figure S2F). Although the markers do not
permit an accurate extrapolation of the sizes of molecules
with unstructured tails, these observations suggest poly(A)
lengths of ≥100 nt.
Polyadenylated mitochondrial tRNAs are rapidly turned over
To investigate the fate of the polyadenylated tRNAs, cells
were incubated in fresh medium for various times, following
EtBr exposure, and sampled periodically for Northern
blotting. After the removal of the drug, and following a short,
EtBr-dose-dependent delay of a few hours, polyadenylated
tRNAs were rapidly degraded, with a half-life of ∼1–
1.5 h (Figure 3A, B). Note that in this, and all
subsequent experiments, tRNASer(UCN) was used as an example
of an extensively polyadenylated tRNA, and, where
appropriate, tRNALeu(UUR) as an example of a less extensively
polyadenylated tRNA. Although some of the
polyadenylated tRNASer(UCN) species may have been trimmed and
repaired, their disappearance was not accompanied by rapid
restoration of the level of mature tRNA (e.g. see Figure 3A),
implying that most of the material was degraded. New
transcription is probably sufficient to account for the gradual
recovery in the level of mature tRNA seen from 16 h following
EtBr removal. Detectable amounts of ND3 mRNA, for
example, which is replenished by new transcription, started to
reappear after approximately the same recovery time
(Supplementary Figure S3B).
For tRNALeu(UUR), which was less affected by EtBr, the
polyadenylated species were also turned over rapidly once
the drug was removed, with the mature tRNA being
restored to its starting level only after 64 h of recovery (Figure
3C, Supplementary Figure S3A). The slow rate of recovery
implies that few, if any, of the polyadenylated tRNAs were
repaired, once EtBr was removed.
Enzymes required for mitochondrial tRNA polyadenylation and turnover
Our observations concerning the polyadenylation of
mitochondrial tRNAs following exposure to EtBr, and their
rapid turnover upon removal of the drug, are
unprecedented. Many questions are raised regarding the
molecular machinery that signals and executes these processes, and
their physiological significance. mtPAP is the only RNA
polymerase inside human mitochondria that has been
previously characterized as being capable of synthesizing
nontemplated poly(A) tails (on mitochondrial mRNAs, (
The enzyme is also responsible for the addition of the
discriminator A during maturation of human mitochondrial
), and is involved in the maturation of
mitochondrial tRNACys in Drosophila (
). The low
proportion of non-A residues (≤1%, Table 1) in the EtBr-induced
tRNA poly(A) tails, similar to that seen in the 3 tails of
mitochondrial mRNAs (
), also suggests that mtPAP is
involved, rather than a less faithful enzyme such as PNPase,
which has been implicated in polyadenylation in
Unusually, mtPAP exhibits relative insensitivity to the
chain terminator cordycepin (3 deoxyadenosine), enabling
its activity to be discriminated from other cellular RNA
). We therefore tested the cordycepin
sensitivity of mitochondrial tRNA polyadenylation produced
by EtBr treatment. At 20 g/ml, cordycepin had no effect
on the polyadenylation of tRNASer(UCN) over 8 h of EtBr
exposure (Figure 4A), despite the fact that 20 g/ml
cordycepin had a profound effect on the steady-state level of ND3
mRNA (Supplementary Figure S4A), synthesis of which
EtBr 5 μg/ml
EtBr 2.5 μg/ml –
+ – + –
– + + – + – +
EtBr 5 μg/ml
EtBr 2.5 μg/ml –
depends on the cordycepin-sensitive mitochondrial RNA
polymerase responsible for global transcription (
contrast, cordycepin treatment for 8 h had little effect on
the steady-state level of tRNASer(UCN) in the absence of EtBr
(Supplementary Figure S4A).
To test further whether mitochondrial tRNA
polyadenylation is due to mtPAP or PNPase, we used siRNA-based
RNA interference combined with EtBr treatment. After
verifying knockdown at the protein level (Supplementary
Figure S4B, S4G), we used Northern blots to examine the
effects on the length of poly(A) tails added to tRNASer(UCN)
(Figure 4B, Supplementary Figure S4C). RNAi directed
against mtPAP consistently resulted in drastic shortening of
the poly(A) tails, but not in their complete abolition
(Figure 4B, Supplementary Figure S4C, E), whereas RNAi
directed against PNPase resulted in tail lengthening (Figure
4B, Supplementary Figure S4C), although this was less
evident when EtBr was used at a lower dose, with RNAi for
less time (Figure 4C, upper panel, Supplementary Figure
S4E). When the two genes were knocked down
simultaneously, tails were slightly decreased in length compared with
untreated cells, or with cells treated with a control siRNA
(Supplementary Figure S4C), consistent with the two
enzymes acting antagonistically.
We took a similar approach to identify the gene
products responsible for turnover of polyadenylated tRNAs
following removal of EtBr. mtPAP knockdown had no
effect on the turnover of the shortened tRNASer(UCN) tails
(Figure 4C, Supplementary Figure S4F), whereas PNPase
knockdown delayed and largely prevented the degradation
of polyadenylated tRNASer(UCN), (Figure 4C, upper panel,
Supplementary Figure S4F), with 3 or 4 days of RNAi
almost completely inhibiting the process. PNPase has been
previously implicated in general RNA turnover in human
mitochondria as a component of the ‘degradosome’ (
We therefore tested the other identified component of this
machinery, the RNA helicase SUV3, again first verifying
the effect of knockdown at the protein level
(Supplementary Figure S4D, G). SUV3 knockdown resulted in
shortened poly(A) tails in the presence of EtBr (Figure 4C, upper
panel, Supplementary Figure S4F), but also inhibited their
subsequent degradation (Figure 4C, upper panel,
Supplementary Figure S4F). To exclude off-target effects, we
confirmed these findings in each case, using a second siRNA
(Supplementary Table S1, Figure S4E, F).
The 2´ phosphodiesterase encoded by PDE12,
previously implicated in the turnover of the poly(A) tails of
human mitochondrial mRNA, in tRNA repair (
) and in
the turnover of specific, oligoadenylated tRNAs (21), was
tested similarly. Although PDE12 knockdown was effective
at the protein level (Supplementary Figure S4D), this had
no effect on the turnover of polyadenylated tRNASer(UCN)
(Figure 4C, lower panel). However, PDE12 knockdown had
reproducible effects on the EtBr-induced adenylation of two
other tRNAs (Supplementary Figure S4H, S4I), that were
previously shown to be susceptible to oligoadenylation in
its absence (
In the case of tRNALys, PDE12 knockdown revealed a
more slowly migrating form of the tRNA that was seen
even prior to exposure to EtBr (blue arrows in
Supplementary Figure S4H), which we assume to correspond with the
previously reported oligoadenylated species (
EtBr exposure, while the accumulation of polyadenylated
species was very similar to that seen in cells treated with
a control siRNA, the mature form of the tRNA was
massively depleted after EtBr treatment, implying that PDE12 is
somehow required for its stabilization, rather than turnover.
The polyadenylated tRNALys species were typically
degraded more slowly during recovery than were those derived
from tRNASer(UCN), but PDE12 knockdown may actually
have accelerated this process. The effects on PNP, mtPAP
and SUV3 knockdown on tRNALys adenylation were
qualitatively similar to those seen for tRNASer(UCN)
(Supplementary Figure S4H).
In the case of tRNAHis, short-tailed molecules were
below the detection limit in PDE12 knockdown cells, prior to
EtBr exposure. However, they were induced by EtBr, with
PDE12 knockdown facilitating the process, such that
almost all of the tRNA was converted to a discrete species
migrating more slowly than in cells treated with a control
siRNA (Supplementary Figure S4I). PDE12 knockdown
did not prevent the turnover of these species during
recovery from EtBr. Once again, knockdown of PNP,
mtPAP and SUV3 produced qualitatively similar effects on
EtBr-induced adenylation and turnover of tRNAHis as seen
for tRNASer(UCN) (Supplementary Figure S4I). The overall
conclusion is that each of the tRNAs tested has a specific
behaviour in response to PDE12 knockdown, EtBr
treatment and recovery. Furthermore, the processes of tRNA
polyadenylation and oligoadenylation, plus the
corresponding deadenylation, appear to operate independently.
Production and turnover of polyadenylated tRNAs depends on mitochondrial metabolism
Macromolecular synthesis is an energy-requiring process,
and the main substrate for polyadenylation is ATP. We
confirmed that inhibition of mitochondrial ATP
synthesis via treatment either with an inhibitor of ATP synthase
(oligomycin, Figure 5A) or an uncoupler (CCCP, Figure 5B,
Supplementary Figure S5A) resulted in a complete block of
the mitochondrial tRNA polyadenylation seen upon EtBr
exposure. CCCP had this effect regardless of whether it was
added prior to or following EtBr exposure (Figure 5B,
Supplementary Figure S5A).
Next we tested the effect of inhibitors of
mitochondrial protein synthesis. Doxycycline prevented
mitochondrial tRNA polyadenylation in response to EtBr, at doses
between 200 and 1000 g/ml (Figure 5C,
Supplementary Figure S5B). Paradoxically, at the highest doses used,
it promoted a tiny accumulation of modified species
comigrating with polyadenylated tRNAs, even in the
absence of EtBr (Figure 5C, Supplementary Figure S5B,
asterisked species). Thiamphenicol had no effect on
mitochondrial tRNASer(UCN) polyadenylation (Figure 5D), while
puromycin had a mild inhibitory effect (Figure 5E), but only
at the highest dose used (10 g/ml). Both drugs had an
inhibitory effect on tRNALeu(UUR) polyadenylation at
somewhat lower doses, i.e. 0.5–1 mg/ml thiamphenicol or 2–5
g/ml puromycin (Supplementary Figure S5C, D).
Doxycycline added during the recovery phase also blocked the
turnover of tRNAs that had been polyadenylated in
response to EtBr (Figure 5F, Supplementary Figure S5E).
This resulted in the accumulation of longer products, both
for tRNASer(UCN) (Figure 5F) and tRNALeu(UUR)
(Supplementary Figure S5E).
To ascertain whether tRNA polyadenylation under these
various conditions was determined only by the extent of
tRNA deacylation, we used acidic polyacrylamide gel-blots,
probed for tRNAs whose aminoacylation status can be
easily visualized by their mobility on such gels, tRNALys and
tRNALeu(UUR) (Figure 6). These tRNAs are also less rapidly
modified than, for example, tRNAPhe or tRNASer(UCN)
(Figures 1 and 2, Supplementary Figures S1A, S2), allowing us
to investigate the acylation status of these tRNAs during a
time when most of the tRNA remained unmodified.
Both tRNAs were extensively deacylated within 3-4 h
of exposure to EtBr (Figure 6A), corresponding roughly
with the period when their polyadenylation started to
become substantial (see Figure 2A, B, Supplementary
Figure S2B). Doxycycline, despite its inhibitory effect on
both polyadenylation (Figure 5C, Supplementary Figure
S5B), and turnover (Figure 5F, Supplementary Figure
S5E), did not abolish deacylation (Figure 6B), merely
delaying it. Thiamphenicol at 200 g/ml, which had no
effect on tRNALeu(UUR) polyadenylation (Supplementary
Figure S5C), also had no effect on deacylation (Figure
6C). Conversely, puromycin, which decreased the extent
of tRNALeu(UUR) polyadenylation (Supplementary Figure
S5D), also inhibited or delayed deacylation (Figure 6D),
although this was pronounced only at the higher dose of the
drug. Overall, we conclude that deacylated mitochondrial
tRNAs can be polyadenylated, following their
A pathological mutant mitochondrial tRNA is polyadenylated
The foregoing data led us to hypothesize that structural
distortion of mitochondrial tRNAs by high levels of EtBr
induced their exclusion from the pool of translationally
competent tRNAs, entraining their polyadenylation and
subsequent degradation. If supported, such a mechanism
may represent a natural quality-control pathway relevant to
EtBr 5 μg/ml
– – – – + – + – + – +
pathology. To test this idea, we made use of a cell line
effectively homoplasmic for the dysfunctional tRNALeu(UUR)
carrying the A3243G MELAS mutation, but in which
normal mitochondrial protein synthesis and respiratory
function is maintained by the presence of a suppressor tRNA
). The A3243G mutation is known to produce a
variety of tRNA abnormalities that effectively cripple its
ability to participate in translation, including decreased
steadystate level and inefficient aminoacylation (
with failure of wobble-base modification (
). The A549
lung-carcinoma-derived cybrid cell line GT, which is almost
(≥99%) homoplasmic for the A3243G mutation but
heteroplasmic for the G12300A tRNALeu(CUN) suppressor
mutation and respiration-competent, was studied under various
conditions facilitating the detection of polyadenylated
mitochondrial tRNAs in control cells.
As in 143B-derived cells (Figures 1–5), tRNASer(UCN) was
polyadenylated in GT cells after treatment with high levels
of EtBr (Figure 7A, upper panel), whereas this was blocked
by treatment with doxycycline. Prolonged (7 h) treatment
of GT cells with a high concentration (1 mg/ml) of
doxycycline in the absence of EtBr also revealed a tiny amount
of modified tRNASer(UCN), co-migrating with the species
polyadenylated after EtBr treatment, essentially the same
as seen in 143B cell cybrids (Figure 5C).
However, tRNALeu(UUR) exhibited a completely different
behaviour in GT cells (Figure 7A, lower panel). EtBr
treatment alone had only a minor effect on the tRNA (Figure
7B, Supplementary Figure S6A), whereas doxycycline
produced a strong, dose-dependent shift in its mobility,
suggestive of polyadenylation, that was partially inhibited by
concomitant treatment with EtBr (Figure 7A, lower panel).
The putatively polyadenylated species of tRNALeu(UUR)
0 d 0.5¨1 2 3 4 5 6 8 24 h EtBr
0 d 1 2 3 4 5 7 25 1 2 3 4 6 24 h TAP
was also faintly visible even in untreated cells. As
observed in 143B cell cybrids (Figure 6B), the deacylation of
tRNALeu(UUR) in control A549 cells that was brought about
by EtBr treatment (Supplementary Figure S6B) was delayed
by prior exposure to doxycycline (Supplementary Figure
S6C), whereas in GT cells, where the (mutant) tRNA was
already partially deacylated, EtBr led to complete
deacylation within 1 h, irrespective of whether or not the cells were
pre-treated with doxycycline (Supplementary Figure S6B,
To confirm that the modification of tRNALeu(UUR)
prominently revealed by doxycycline treatment in GT cells was
once again 3 polyadenylation, we analyzed the products
by Sanger sequencing of the prominent modified band after
circularization and cloning, which was once again followed
up by tNGS (Table 2, Supplementary Table S3). Tails were
again composed primarily of poly(A), with non-A residues
at a similar frequency as in the tailed tRNALeu(UUR) species
generated in response to EtBr treatment. In this case,
however, most of the mutant tRNA retained CCA.
In this study, we followed up the unexpected observation
that, at high doses, EtBr treatment of cultured human
cells resulted in the polyadenylation of mitochondrial
tRNAs and their subsequent degradation after removal of the
drug. Other cellular RNAs, such as a cytosolic tRNA or
a mitochondrial mRNA, were not modified in this way by
EtBr. Mitochondrial tRNA polyadenylation was dependent
on ATP synthesis and required mtPAP and SUV3, while
turnover required PNPase and SUV3, and inhibitor studies
suggested the possible involvement of the mitoribosome. A
dysfunctional and structurally abnormal tRNA associated
with human disease was also subject to polyadenylation,
implying that the pathway may operate as a more general
quality-control mechanism. Our findings are summarized
in Figure 8.
Physiological relevance of tRNA polyadenylation
As discussed in the Introduction, polyadenylation serves
diverse purposes in biology. In metazoan mitochondria,
mRNA polyadenylation remains enigmatic, with poly(A)
apparently serving as a tag for stabilization of some RNAs,
but the destabilization of others. Oligoadenylated, mostly
truncated mitochondrial tRNA species have been
previously reported (
), and have been observed to
accumulate when PDE12 was knocked down (
). While some
have been proposed as processing intermediates (
overall provenance and abundance remain unexplained.
In the present study, polyadenylation of
mitochondrial tRNAs was revealed under the admittedly
nonphysiological conditions of prolonged treatment of cells
with a nucleic acid intercalating agent, EtBr. However,
it was also observed in the case of a tRNA carrying
a pathological mutation which impairs base-modification
and aminoacylation, and disables translational function. In
both cases, polyadenylated tRNAs accumulated to
substantial levels, but only while their turnover was blocked,
respectively by EtBr itself, or by doxycycline. Treatment with a
lower concentration of EtBr, sufficient to block
drial transcription, did not result in tRNA polyadenylation
), but even the higher level of EtBr did not impair cell
viability (Supplementary Figure S1C). Since the affinity of
EtBr for structured RNA is less than for DNA, our findings
are consistent with the idea that tRNA polyadenylation is
induced by structural distortions resulting either from EtBr
intercalation or from mutation, rendering the tRNA
structurally abnormal and impairing its physiological function.
The highly variable susceptibility to polyadenylation in the
presence of EtBr, exhibited by different mitochondrial
tRNAs, most likely reflects the different degree of structural
distortion produced by the drug. Whether RNA
polyadenylation depends on exact structural features, or is a default
pathway for any free 3 end of a structurally abnormal or
misfolded RNA remains to be determined. The latter seems
probable, if it is assumed that this operates as a surveillance
The biogenesis of tRNAs is a complex multi-step process,
hence it is logical that any significant failure thereof that
impairs translational function would be subject to a
qualitycontrol process geared to preventing interference with the
fidelity or efficiency of protein synthesis, as previously
A3243G mutant Leu(UUR)
ferred in bacteria (
). Based on our study, tRNAs appear
to be the only class of mitochondrial transcript that are
systematically destabilized by polyadenylation, in response to
structural aberrations. This pathway may represent the
original function for poly(A) in mitochondria, with the addition
of stable poly/oligo(A)-tails at the 3 ends of mRNAs and
rRNAs having evolved later, as a distinct process. It may
also serve a wider function, for example, to eliminate
Although RNA-intercalating drugs are not frequently
encountered in nature, structural abnormalities in tRNAs
will result from many other causes, including pathological
mutations, errors in RNA processing or base modification,
or the actions of some antibiotic drugs (
). All such
processes are potentially influenced by exposure to diverse
environmental agents and conditions. Point mutations in tRNA
genes are the most common class of pathological lesion in
mtDNA after deletions (which themselves affect multiple
tRNAs). Many of them have multiple downstream effects
on tRNA structure, abundance, acylation and translational
function, while another prominent class of disease
mutations affects the nuclear genes responsible for these various
steps in mitochondrial tRNA metabolism (
Polymorphisms that do not result in an overt pathology might also
give rise to tRNAs that are poorly acylated, modified or
processed in, and depend on this pathway for the maintenance
of translational fidelity.
RNA surveillance based on polyadenylation may also
function as defence against the mitochondrial milieu being
invaded or hijacked by a virus (
), or as a response to the
hypothetical case of cytosolic RNAs that might have been
aberrantly imported into mitochondria, disturbing
mitochondrial functions. Programmed import of specific
cytosolic tRNAs has been documented in many organisms (
Machinery of mitochondrial tRNA polyadenylation and turnover
The relative cordycepin insensitivity of EtBr-induced
mitochondrial tRNA polyadenylation (Figure 4A) rules out
most cellular RNA polymerases from involvement,
including the mitochondrial RNA polymerase and the
major polyadenylases of the nucleus and cytosol. Conversely,
RNA interference (Figure 4B, C, Supplementary Figure
S4C, E) clearly implicates mtPAP (PAPD1), which was
already suggested by the low incorporation of non-A residues
into tRNA 3 tails (Tables 1 and 2), similar to that seen in
mitochondrial mRNAs (
). A low rate of incorporation of
non-A residues is consistent with the fact that mtPAP is able
to use all four ribonucleoside triphosphates in vitro (
furthermore, has been implicated in the oligouridylation of
histone mRNAs in the cytoplasm (
), although this has
been questioned (
). Incorrectly processed transcripts
have also been reported to be uridylated in mitochondria
). mtPAP knockdown did not completely abolish the
modification of mitochondrial tRNAs upon EtBr
treatment. It consistently resulted in the appearance of tRNA
species extended by only a short distance (Figure 4B, C,
Supplementary Figure S4E), compared with the longer tails
seen when mtPAP was still present at wild-type levels.
Although we cannot exclude that this reflects a small amount
of residual mtPAP activity, it may also indicate the action
of another, unidentified oligoadenylase, as suggested also
in the case of mitochondrial mRNAs (
). There are no
obvious candidates for such an enzyme, but one possibility
may be the 3 nucleotidyl transferase (TRNT1) that creates
the CCA terminus of both cytosolic and mitochondrial
tRNAs. This enzyme is part of a superfamily (68) that also
includes the poly(A) polymerases (
). However, testing its
involvement in tRNA adenylation is not straightforward,
because of its pleiotropic functions in tRNA metabolism.
EtBr-induced tRNA polyadenylation appears also to
depend on the SUV3 helicase, since its knockdown resulted in
the shortening of tRNA poly(A) tails (Figure 4C,
Supplementary Figure S4F). SUV3 in different taxa has already
been shown to play diverse roles in mitochondrial RNA
metabolism. The SUV3 orthologue in Schizosaccharomyces
pombe has been implicated in mRNA processing (
Drosophila it functions in the processing of mitochondrial
tRNAs and in poly(A) tail-lengthening of 16S rRNA and of
some mitochondrial mRNAs (
). Furthermore, SUV3 has
previously been reported to facilitate the polymerase
function of human mtPAP, under specific conditions, involving
a direct interaction between the proteins (
). We surmise
that the highly structured nature of tRNAs leads to
transient interactions of nascent poly(A) tails with the body of
the tRNA, impeding the processivity of mtPAP, and that
SUV3 can relieve this constraint. This may apply, for
example, in the case of tRNASer(UCN), which has a
pyrimidine(and specifically U-) rich region near to its 3 end, although
this structure may also contribute to the rather efficient and
stable polyadenylation of the tRNA.
In Saccharomyces cerevisiae, Suv3 is a component of the
mitochondrial ‘degradosome’, the enzyme complex
responsible for RNA turnover (
). Human SUV3 (SUPV3L1) has
been inferred to function similarly (
), but in combination
with a different nuclease, PNPase instead of Dss1 (
despite the fact that most PNPase is located in the
intermembrane space (75). In our analysis, SUV3 knockdown
also blocked the turnover of polyadenylated mitochondrial
tRNA. Thus, SUV3 appears to be required both for full
polyadenylation and for turnover.
Knockdown of PNPase resulted in tRNA poly(A) tail
lengthening (Figure 4B, Supplementary Figure S4C, E) as
well as inhibition of turnover (Figure 4C, Supplementary
Figure S4F). PNPase is considered to operate reversibly, i.e.
it can function, effectively, either as an RNA polymerase
or as a nuclease, although catalyzing a different chemistry
than conventional enzymes of either class. Its role in
creating poly(A) tails in chloroplasts has already been
mentioned, but in human mitochondria its role is generally
antagonistic to mtPAP (
), and this appears to be
the case here also for tRNA polyadenylation, with mtPAP
required for tail lengthening but PNPase performing the
opposite role, as well as being required for the (delayed)
degradation of the polyadenylated tRNAs, following removal
of EtBr. The PDE12-mediated removal of oligo(A) tails
from a subset of tRNAs (including tRNAHis and tRNALys;
21) appears to be a largely independent process, in the
sense that PNPase knockdown did not affect the turnover
of oligoadenylated tRNAs, while PDE12 knockdown did
not block the degradation of those bearing longer poly(A)
tails (Supplementary Figure S4H, I). However, EtBr
treatment induced both the oligoadenylation and
polyadenylation of these tRNAs. Interestingly, different tRNAs also
varied in their susceptibility to truncation at CCA prior
to polyadenylation, with tRNASer(UCN) and the A32343G
mutant tRNALeu(UUR) both showing a very low amount of
truncation compared with other tRNAs (Tables 1 and 2).
Although polyadenylation of structurally abnormal
tRNAs might be considered a tag for their destruction, it is
perhaps more parsimonious to regard polyadenylation by
mtPAP as a default pathway for misfolded RNAs, which
also entrains their PNPase/SUV3-mediated degradation by
virtue of the unstructured nature of the poly(A) tail. If
assumed to be an ancient process, it is curious that
poly(A)mediated RNA turnover is absent from S. cerevisiae
mitochondria, which lack orthologues for both mtPAP and
PNPase. Thus, if there is an analogous pathway in yeast
mitochondria for removal of structurally abnormal tRNAs, it
must involve other enzymes, for which the nuclease Dss1
) is one obvious candidate.
Possible involvement of the translational machinery in tRNA polyadenylation
The varying effects of different inhibitors of
mitochondrial protein synthesis on these processes suggest a
possible but not straightforward association with the
translational machinery. Doxycycline strongly inhibited
EtBr-induced tRNA polyadenylation and the subsequent
turnover of polyadenylated tRNAs, whereas thiamphenicol
and puromycin had only minor effects. In contrast,
doxycycline enhanced the polyadenylation of the A3243G-mutant
tRNALeu(UUR) (Figure 7A), which occurred in the absence
of EtBr, despite the fact that the mutant tRNA probably
participates only minimally in protein synthesis.
By analogy with data on bacterial ribosomes, doxycycline
blocks aminoacyl-tRNA access to the ribosome A-site, by
binding to a region which overlaps the binding pocket for
the tRNA anticodon stem-loop (
). This prevents the
first step in the elongation cycle ((80); see (
) for review).
However, thiamphenicol, like its less potent analogue
chloramphenicol, inhibits the peptidyl transferase step of
elongation (83), although this depends on the specific terminal
amino acids in the nascent peptide chain and the A-site (
Puromycin acts as a chain terminator (
) of both cytosolic
and mitochondrial translation. The different effects of these
drugs suggests that tRNA polyadenylation and turnover
might be brought about by a machinery associated with the
mitoribosome A-site, or modulated by a signal originating
there and reflecting the manner of its occupancy. However,
EtBr itself abrogated mitochondrial protein synthesis
(Supplementary Figure S1H), while doxycycline did not induce
a significant accumulation of polyadenylated tRNAs except
at very high levels, or of a structurally abnormal tRNA that
was already present. The effects of doxycycline could
therefore be secondary, or even independent of the translation
The components of the human mitochondrial
‘degradosome’, SUV3 and PNPase (
), have been reported to
co-localize in discrete foci that contain other components
of the machinery of mitochondrial RNA processing,
including mtPAP (
), as well as being the sites of
mitoribosome biogenesis (
). A sub-population of these foci
is intimately associated with mtDNA nucleoids (
However, translation initiation and elongation factors have
not been reported as components of these granules (86),
implying that they are not sites of ongoing protein
synthesis, although affinity purification of the mitochondrial
ribosome recycling factor mtRRF did result in the
coisolation of nucleoid, RNA processing and translation
). Cross-linking studies have also revealed such
). Some other RNA quality control pathways,
such as nonsense-mediated decay or no-go decay, are clearly
). Furthermore, Temperley et al.
) found that deadenylation of an abnormal ATP8/ATP6
mRNA, produced as a result of a mutation at the gene
boundary with MTCO3, was dependent on ongoing
translation, although they did not determine whether the
degradation machinery was directly associated with the
mitoribosome. In sum, the involvement of the mitoribosome in
polyadenylation and turnover of abnormal tRNAs is
unproven. The presence of an invalid tRNA in the
mitoribosome A-site may be involved in triggering the response, but
tRNA surveillance may also operate independently of the
Significance for mitochondrial tRNA pathologies
The existence of a surveillance system to detect and dispose
of abnormally structured RNAs, including the pathological
mutant tRNALeu(UUR) bearing the A3243G mutation,
suggests the importance of this pathway in human pathology.
The molecular, cellular and physiological effects of
pathological mutations affecting mitochondrial tRNAs are highly
diverse, and exhibit a great variety of tissue specificities that
remain largely unexplained. Mostly, they are marked by
decreased steady-state abundances of the affected tRNA.
Failure of a surveillance mechanism for removing
structurally abnormal tRNAs could result in their participating
in protein synthesis, triggering harmful downstream effects.
This might explain some puzzling observations: for
example, the case of a carrier mother homoplasmic for a
pathological mutation in mitochondrial tRNAVal that is present
in her cells at very low levels without drastic clinical
effects, yet is robustly associated with fetal/infantile lethality
in her children (
). The high heteroplasmic threshold
levels for pathological tRNA mutations also makes sense if
aberrant tRNAs are disposed of before they can cause
active damage. Finally, the broad failure of negative selection
against tRNA mutations in the female germline (95) could
be accounted for by a stringent surveillance mechanism that
maintains aberrant tRNAs only at very low levels.
The polyadenylation and turnover of abnormal tRNAs
may be potentiated by more general stress responses. The
possible involvement of the mitoribosome has already
been discussed, but other physiological triggers elicited by
high levels of EtBr (Supplementary Figure S1C–G) may
be involved, including increased ROS, respiratory defects
and mtDNA copy number depletion. Activating a
surveillance pathway targeting abnormal RNAs in affected tissues
may be one fruitful approach to combating mitochondrial
The precise structural distortions that trigger
polyadenylation of deacylated tRNAs and their subsequent
degradation are not obvious, and may depend on further, thus far
unidentified factors. The response of the mitoribosome to
very low levels of a given tRNA has not been studied
extensively, but prolonged stalling is assumed to trigger
premature termination and ribosome recycling. Four members of
the (class 1) ribosomal release factor family have been
identified in mitochondria (
), three of which have been
proposed to be involved in rescuing stalled mitoribosomes (
). One or more of them could be involved in the response
to tRNA insufficiency. Limiting their action in the event of
high levels of heteroplasmy for a pathological tRNA
mutation could be one way to break a futile cycle of
mitoribosome initiation and premature termination, enabling the
productive synthesis of mitochondrially encoded
polypeptides to proceed.
Supplementary Data are available at NAR Online.
We thank Eeva-Marja Turkki for library preparation and
sequencing, Laurence Bindoff, Bob Lightowlers, Brendan
Battersby and Uwe Richter for advice and useful
discussions, Maarit Partanen for technical assistance and Troy
Faithfull for critical reading and editing of the manuscript.
Academy of Finland [Centre of Excellence grant 272376;
Academy Professorship grant 283157 to H.T.J.];
University of Tampere; Tampere University Hospital Medical
Research Fund; Sigrid Juselius Foundation. Funding for open
access charge: University of Tampere.
Conflict of interest statement. None declared.
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