mRNA localization to the mitochondrial surface
allows the efficient translocation inside the organelle
of a nuclear recoded ATP6 protein
VALE´RIE KALTIMBACHER,1CRYSTEL BONNET,1GAE¨LLE LECOEUVRE, VALE´RIE FORSTER,
JOSE´-ALAIN SAHEL, and MARISOL CORRAL-DEBRINSKI
Laboratoire de Physiopathologie Cellulaire et Mole ´culaire de la Re ´tine, INSERM U592 and Universite ´ Pierre et Marie Curie (UPMC-Paris6),
Ho ˆpital St. Antoine, 75571 Paris, Cedex 12 France
As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the
mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 39UTR. This phenomenon is
conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is
not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence
and the 39UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The
ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V.
In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface
in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the
cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 39UTR of the SOD2
gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial
surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the
mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity.
Keywords: mRNA sorting to the mitochondrial surface; mitochondrial cotranslational import; nuclear encoded ATP6 gene
A key feature of eukaryotic cells is their organization into
separate subcellular compartments, each containing dis-
tinct sets of proteins. The sorting of several proteins
destined to organelles involves mRNA localization. This
specific localization might be preferable to protein locali-
zation; indeed, one mRNA molecule can serve as a template
for multiple rounds of translation. Thus, localizing an
mRNA rather than the protein to its site of action offers
obvious advantages (Jansen 2001; Tekotte and Davis 2002).
Mitochondria occupy a central position in the overall
metabolism of eukaryotic cells; hence, the oxidative phos-
phorylation (OXPHOS), the Krebs’s cycle, the urea cycle, the
heme biosynthesis, and the fatty acid oxidation take place
within the organelle. Mitochondrial biogenesis is a complex
process that requires the concerted expression of both
nuclear and mitochondrial genomes. More than 99% of
mitochondrial proteins are encoded by the nucleus and
synthesized in the cytoplasm. Mitochondrial sorting of
mRNAs encoding mitochondrial proteins might likely rep-
resent a key step to ensuring the functionality of the
corresponding polypeptides inside the organelle. In this case,
a cotranslational phase might assist the import of the
precursors (Corral-Debrinski et al. 1999, 2000; Fu ¨nfschilling
and Rospert 1999; George et al. 2002). We have demon-
strated that in yeast, 47% of mRNAs encoding mitochon-
drial proteins are transported to the organelle surface
(Sylvestre et al. 2003b). Among them, ATP2 mRNA, encoding
the b-subunit of ATP synthase, exclusively localizes to the
mitochondrial surface. Two sequences within the ATP2
1These authors contributed equally to this work.
Reprint requests to: Marisol Corral-Debrinski, Laboratoire de Physi-
opathologie Cellulaire et Mole ´culaire de la Re ´tine, INSERM U592 and
Universite ´ Pierre et Marie Curie (UPMC-Paris6), Ho ˆpital St. Antoine–Ba ˆt.
Kourilsky 184, rue du Fbg. Saint-Antoine 75571 Paris, Cedex 12 France;
e-mail: firstname.lastname@example.org; fax: +33-1-49-28-66-63.
Article published online ahead of print. Article and publication date are
RNA (2006), 12:1408–1417. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2006 RNA Society.
transcript are involved in its specific subcellular local-
ization—the region coding for the mitochondrial targeting
sequence (MTS) and the 39 untranslated region (39UTR).
Remarkably, the in vivo impairment of ATP2 mRNA
sorting, due to the absence of its 39UTR, is associated with
a severe respiratory deficiency. This deficiency is the
consequence of an inefficient import of the precursor,
indicating that mRNA targeting to the surface of mito-
chondria is essential to ensure this process (Margeot et al.
2002). Interestingly, we demonstrated that several mRNAs
encoding mitochondrial proteins preferentially localized to
the mitochondrial surface in HeLa cells (Sylvestre et al. 2003a).
We have addressed the question of whether a protein that is
normally encoded by mitochondrial DNA (mtDNA) can be
efficiently translocated inside the mitochondria by the use
of signals that will address its mRNA to the surface of the
organelle. The rationale behind this is to allow a cotransla-
tional import mechanism. Thus, the precursor will be
maintained in an import-competent conformation, imped-
ing its aggregation before or during translocation through
the TOM (translocase of the outer membrane) and TIM
(translocase of the inner membrane) import complexes. The
strategy of expressing mtDNA-encoded genes in the nuclear-
cytoplasmic compartment is called allotopic expression.
Up until today, when tried in mammalian cells, allotopic
expression did not lead to an efficient mitochondrial trans-
location of precursors examined, probably due to the high
hydrophobic nature of polypeptides encoded by mtDNA
(Oca-Cossio et al. 2003; Smith et al. 2004).
We associated the MTS and the 39UTR of the nuclear
gene SOD2 to a re-engineered nucleus-localized ATP6 gene.
SOD2 codes for an intramitochondrial free radical scaveng-
ing enzyme, which is the first line of defense against
superoxide produced as a byproduct of OXPHOS (Li et al.
1995). We have previously shown that SOD2 mRNA local-
izes to the mitochondrial surface in human cells (Sylvestre
et al. 2003a). Furthermore, a recent report described that in
HeLa cells, SOD2 mRNA is associated to the mitochondrial
surface, via its 39UTR linked to the K homology domain of
A-kinase anchor protein 121, AKAP121 (Ginsberg et al.
2003). HeLa cells were stably transfected with two different
plasmids. One contains the MTS representing the first 30
amino acids of the protein in frame with the AUG codon of
the recoded ATP6 gene, the other combines both the MTS
and 39UTR. We were able to demonstrate that (1) both
hybrid ATP6 mRNAs localize to the mitochondrial surface,
especially the one possessing the two targeting signals; (2)
fusion protein synthesized from each construction localizes
to mitochondria in vivo; (3) precursor forms of the ATP6
protein translated in the cytoplasm are successfully trans-
located inside the organelle. Interestingly, the presence of
both mRNA-targeting signals leads to a 1.8-fold increase in
the amount of fully mitochondrial imported protein com-
pared with the presence of the MTS alone. Therefore, the
strategy of directing a hybrid mRNA to the mitochondrial
surface allows the mitochondrial import of the nucleus-
encoded ATP6 gene in a highly efficient way.
Construction of the re-engineered mitochondrial
To accomplish allotopic expression of the ATP6 gene
originally located in the mitochondrial genome, we synthe-
sized the full-length nuclear version of the gene-converting
codons AUA to AUG and codons UGA to UGG to ensure
the accurate translation of the transcript by cytoplasmic
ribosomes. Eleven alterations were performed in the ATP6
ORF by in vitro direct mutagenesis. To force the localiza-
tion of the recoded ATP6 mRNA to the mitochondrial
surface, we associated with the ORF the cis-acting elements
involved in the mitochondrial sorting of the SOD2
mRNA—the sequence coding for the MTS and the 39UTR
(as described in the Materials and Methods section).
Consequently, we obtained two different constructions
cloned in the pCMV-Tag 4A vector that we named,
ATP6-39UTRSOD2. The SOD2MTSATP6-39UTRSV40contains
the sequence encoding the first 30 amino acids of SOD2 in
frame with the AUG codon of the recoded ATP6 gene. In
this plasmid, the SV40 poly(A) signal functions as the
39UTR. In the SOD2MTSATP6-39UTRSOD2vector, both
the MTS and the 39UTR of SOD2 were associated with
the ATP6 ORF. The 39UTR of 215 bp (Ginsberg et al. 2003)
was inserted at the end of the ORF following the Flag
epitope and replacing the SV40 poly(A) signal.
Subcellular distribution of hybrid ATP6 mRNAs in
39UTRSV40or SOD2MTSATP6-39UTRSOD2vectors were
examined to determine the steady-state levels of hybrid
ATP6 mRNAs. Total RNAs were extracted from four in-
dependent populations of stably transfected cells (S.T 1 and
S.T 2 for SOD2MTSATP6-39UTRSV40; S.T 3 and S.T 4 for
SOD2MTSATP6-39UTRSOD2). Figure 1A illustrates results
obtained after RT–PCR analyses. The abundance of endog-
enous ATP6, COX6c, and SOD2 transcripts as well as
hybrid ATP6 mRNAs was examined using conditions
described in Table 2 (below). No major differences in
the amount of the SOD2MTSATP6 mRNA were observed in
cell lines expressing either SOD2MTSATP6-39UTRSV40or
To examine the ability of SOD2 signals associated with
the recoded ATP6 gene to direct hybrid mRNAs to the
mitochondrial surface, we determined their subcellular
localization in the four stable cell lines obtained. For this
cells transfectedwith eitherSOD2MTSATP6-
Mitochondrial targeting of a recoded ATP6 protein
expected sizes of the PCR products, the quantity of RNA used for
reverse-transcription, and the number of PCR cycles performed.
Densitometric analyses (Quantity One, Bio-Rad software) were
performed the amount of both hybrid ATP6 and SOD2 transcripts
in either mitochondrion-bound polysomes or free-cytoplasmic
polysomes. Three independent RNA preparations from M-P and
F-P fractions were subjected three times to RT–PCR analyses.
We are grateful to Dr. Yu Chun Lone for useful discussions and
comments on the manuscript and to Dr. Gonzalo M. Claros for the
mitochondrial import ability predictions and useful discussions. This
work was supported by funds from the INSERM (U 592), the French
associations AFM (grant MNM 2004 and 2005), FRM, and Retina.
V.K. is the recipient of a French government fellowship, and C.B., of
a Fe ´de ´ration des Aveugles et Handicape ´s Visuels de France’s award.
Received January 16, 2006; accepted April 13, 2006.
Claros, M.G. and Vincens, P. 1996. Computational methods to
predict mitochondrially imported proteins and their transit
peptides. Eur. J. Biochem. 241: 779–786.
Claros, M.G., Perea, J., Shu, Y., Samatey, F.A., Popot, J.-L., and
Jacq, C. 1995. Limitations to in vivo import of hydrophobic
proteins into yeast mitochondria: The case of a cytoplasmic
synthesized apocytochrome b. Eur. J. Biochem. 228: 762–771.
Corral-Debrinski, M., Belgareh, N., Blugeon, C., Claros, M.G.,
Doye, V., and Jacq, C. 1999. Overexpression of yeast karyopherin
Pse1p/Kap121p stimulates the mitochondrial import of hydro-
phobic proteins in vivo. Mol. Microbiol. 31: 1499–1511.
Corral-Debrinski, M., Blugeon, C., and Jacq, C. 2000. In yeast, the 39
Untranslated Region or the presequence of ATM1 is required for
the exclusive localization of its mRNA to the vicinity of mito-
chondria. Mol. Cell. Biol. 20: 7881–7892.
DiMauro, S. 2004. Mitochondrial medicine. Biochim. Biophys. Acta
Fu ¨nfschilling, U. and Rospert, S. 1999. Nascent polypeptide-associated
complex stimulates protein import into yeast mitochondria.
Mol. Biol. Cell 10: 3289–3299.
George, R., Walsh, P., Beddoe, T., and Lithgow, T. 2002. The nascent
polypeptide-associated complex (NAC) promotes interaction of
ribosome with the mitochondrial surface in vivo. FEBS Lett. 516:
Ginsberg, M.D., Feliciello, A., Jones, J.K., Avvedimento, E.V., and
Gottesman, M.E. 2003. PKA-dependent binding of mRNA to the
mitochondrial AKAP121 protein. J. Mol. Biol. 327: 885–897.
Guy, J., Qi, X., Pallotti, F., Schon, E.A., Manfredi, G., Carelli, V.,
Martinuzzi, A., Hauswirth, W.W., and Lewin, A.S. 2002. Rescue of
a mitochondrial deficiency causing Leber Hereditary Optic Neu-
ropathy. Ann. Neurol. 52: 534–542.
Jansen, R.-P. 2001. mRNA localization: Message on the move. Nat.
Rev. Mol. Cell Biol. 2: 247–256.
Joshi, S. and Burrows, R. 1990. ATP synthase complex from bovine
heart mitochondria. J. Biol. Chem. 265: 14518–14525.
Karniely, S. and Pines, O. 2005. Single translation–dual destination:
Mechanisms of dual protein targeting in eukaryotes. EMBO Rep.
Kloc, M., Zearfoss, N.R., and Etkin, L.D. 2002. Mechanisms of
subcellular mRNA localization. Cell 108: 533–544.
Li, Y.H., Huang, T.-T., Carlson, E.J., Melov, S., Ursell, P.C.,
Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H.,
et al. 1995. Dilated cardiomyopathy and neonatal lethality in
mutant mice lacking manganese superoxide dismutase. Nature
Genet. 11: 376–381.
Manfredi, G., Fu, J., Ojaimi, J., Sadlock, J.E., Kwong, J.Q., Guy, J., and
Schon, E.A. 2002. Rescue of a deficiency in ATP synthesis by
transfer of MTATP6, a mitochondrial DNA-encoded gene to the
nucleus. Nat. Genet. 30: 394–399.
Margeot, A., Blugeon, C., Sylvestre, J., Jacq, C., and Corral-
Debrinski, M. 2002. In Saccharomyces cerevisiae, ATP2 mRNA
sorting to the vicinity of mitochondria is essential for respiratory
function. EMBO J. 21: 6893–6904.
Mariottini, P.C.A., Riley, M., Cottrell, B., Doolittle, R.F., and
Attardi, G. 1986. Identification of the polypeptides encoded in
the unassigned reading frames 2, 4, 4L, and 5 of human
mitochondrial DNA. Proc. Natl. Acad. Sci. 88: 1563–1567.
Nagley, P., Farrell, L.B., Gearing, D.P., Nero, D., Meltzer, S., and
Devenish, R.J. 1995. Assembly of functional proton-translocating
ATPase complex in yeast mitochondria with cytoplasmically
synthesized subunit 8, a polypeptide normally encoded within
the organelle. Proc. Natl. Acad. Sci. 85: 2091–2095.
Oca-Cossio, J., Kenyon, L., Hao, H., and Moraes, C.T. 2003.
Limitations of allotopic expression of mitochondrial genes in
mammalian cells. Genetics 165: 707–720.
Owen, R., Lewin, A.P., Peel, A., Wang, J., Guy, J., Hauswirth, W.W.,
Stacpoole, P.W., and Flotte, T.R. 2000. Recombinant Adeno-
associated virus vector-based gene transfer for defects in oxidative
metabolism. Hum. Gene Ther. 11: 2067–2078.
Shalgi, R., Lapidot, M., Shamir, R., and Pilpel, Y. 2005. A catalog of
stability-associated sequence elements in 39UTRs of yeast mRNAs.
Genome Biol. 6: R86.
Smeitink, J., van den Heuvel, L., and DiMauro, S. 2001. The genetics
and pathology of oxidative phosphorylation. Nat. Genet. 342: 342–
Smith, P.M., Ross, G.F., Taylor, R.W., Turnbull, D.M., and
Lightowlers, R.N. 2004. Strategies for treating disorders of the
mitochondrial genome. Biochim. Biophys. Acta 1659: 232–239.
Sylvestre, J., Margeot, A., Jacq, C., Dujardin, G., and Corral-
Debrinski, M. 2003a. The role of the 39 untranslated region in
mRNA sorting to the vicinity of mitochondria is conserved from
yeast to human cells. Mol. Biol. Cell 14: 3848–3856.
Sylvestre, J., Vialette, S., Corral-Debrinski, M., and Jacq, C. 2003b.
Long mRNAs coding for yeast mitochondrial proteins of pro-
karyotic origin localize to the vicinity of mitochondria. Genome
Biol. 4: R44.
Tekotte, H. and Davis, I. 2002. Intracellular mRNA localization:
Motors move messages. Trends Genet. 18: 636–642.
Valnot, I., von Kleist-Retzow, J.C., Barrientos, A., Gorbatyuk, M.,
Taanman, J.W., Mehaye, B., Rustin, P., Tzagoloff, A., Munnich, A.,
and Rotig, A. 2000. A mutation in the human heme A:farnesyl-
transferase gene (COX10) causes cytochrome c oxidase deficiency.
Hum. Mol. Genet. 9: 1245–1249.
Verner, K. 1993. Co-translational protein import into mitochondria:
An alternative view. Trends Biochem. Sci. 18: 364–371.
Zeviani, M. and Carelli, V. 2003. Mitochondrial disorders. Curr. Opin.
Neurol. 16: 585–594.
Mitochondrial targeting of a recoded ATP6 protein