Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

Biochimie xxx (2013) 1e8

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Research paper

Targeting of cytosolic mRNA to mitochondria: Naked RNA can bind to the mitochondrial surface Morgane Michaud, Laurence Maréchal-Drouard, Anne-Marie Duchêne* Institut de Biologie Moléculaire des Plantes, UPR 2357 du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 July 2013 Accepted 8 November 2013 Available online xxx

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: mRNA sorting mRNA localization Protein targeting Mitochondria TOM VDAC Plant

1. Introduction Mitochondria are essential organelles that play a fundamental role in energy production. They also perform many essential functions in metabolism of nucleotides, amino acids, lipids, vitamins and cofactors, and participate in apoptosis. Their dysfunctions are linked to numerous pathologies in human [1], and to the plant cytoplasmic male sterility character used in breeding technologies [2,3]. Mitochondria originate from the endosymbiosis of an alphaproteobacterium. Over the course of evolution, most of the bacterial genes have been lost or transferred to the nucleus. Thus, to build the active mitochondrion, most of proteins (more than 1000) and numerous RNAs need to be imported from the surrounding cytosol [4]. Protein import into mitochondria involves the cooperation of different complexes in the outer (OM) and the inner (IM) mitochondrial membranes. Proteins first interact with the OM, and

Abbreviations: GAPDH, glyceraldehyde-3-P-dehydrogenase; MDH, malate dehydrogenase; MTS, mitochondrial targeting sequence; NAD9, NADH dehydrogenase subunit 9; PDH-E1, pyruvate dehydrogenase E1; RPL12-p, plastidial ribosomal L12 protein; TOM, Translocase of the Outer Mitochondrial membrane; VDAC, voltage dependent anion channel; UTR, unstranslated region. * Corresponding author. Tel.: þ33 367155369; fax: þ33 388614442. E-mail address: [email protected] (A.-M. Duchêne).

nearly all mitochondrial proteins have to pass through TOM complex (Translocase of the Outer Mitochondrial membrane) before reaching their final destination in mitochondria. Docking to the OM and translocation through the mitochondrial membranes are dependent on amino acid sequences within the protein, and the most classical signal, called MTS, is located at the N-terminus of the protein. Two models, which are not exclusive, have been described for the first steps of import. The widely accepted one is a posttranslational import. Protein are translated on free cytosolic polysomes, then targeted and imported into mitochondria. In accordance with this model, in vitro systems show that most proteins can be imported post-translationally into isolated mitochondria. Another model is emerging in favour of a co-translational import. In agreement with this view, cytosolic polysomes are found associated with the mitochondrial surface [5,6] and numerous cytosolic mRNAs encoding mitochondrial proteins are detected in mitochondrial fractions in yeast, plants or animals [7e11]. Sequences within the mRNA molecules have been shown to be essential for targeting. These cis-elements are localized in the coding sequence or in the 30 untranslated region (30 UTR). Up to now only a few transacting factors involved in mRNA targeting have been identified in yeast, such as the RNA binding protein Puf3p or subunits of TOM complex [12e14]. Messenger RNA sorting to the mitochondrial surface has been demonstrated in potato [9]. The differential localization of about 20

0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.11.007

Please cite this article in press as: M. Michaud, et al., Targeting of cytosolic mRNA to mitochondria: Naked RNA can bind to the mitochondrial surface, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.11.007

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M. Michaud et al. / Biochimie xxx (2013) 1e8

cytosolic mRNAs was analysed by RT-qPCR in mitochondria versus total fractions, and a 10 fold gradient was observed between the less and the most mitochondrial associated mRNAs. The most targeted mRNAs coded for mitochondrial associated proteins such as MDH (Malate dehydrogenase), a matrix enzyme involved in TCA cycle, or GAPDH (Glyceraldehyde-3-P-dehydrogenase), a glycolytic enzyme associated to OM [15,16]. The less associated mRNAs coded for dual-targeted plastidial-mitochondrial proteins (seryl-tRNA synthetase, prolyl-tRNA synthetase) and for a plastidial ribosomal protein (RPL12-p). The mechanisms allowing mitochondrial localization of mRNAs are not known in plants. Nor the cytosolic factors involved in the mitochondrial targeting of mRNAs, neither the mitochondrial receptors responsible for the docking of mRNAs have been identified. In this paper, we develop an in vitro system to study mRNA binding to the mitochondrial surface. We show that mRNAs are able to bind to isolated mitochondria without the need of ribosomes or cytosolic factors, and our results strongly suggest that mRNA docking to the OM requires at least one component of TOM complex.

2. Materials and methods 2.1. Mitochondria preparation Mitochondria are extracted from potato tubers (cv Bintje) according to Michaud et al. [9] in buffers supplemented with 10 mM MgCl2. After differential centrifugations and purification through a sucrose gradient, mitochondria are finally resuspended at the concentration of 20 mg proteins/ml in washing buffer (sucrose 0.3 M, potassium phosphate pH 7.5 10 mM, glycine 5 mM) complemented with 10 mM MgCl2 or 1 mM EDTA depending of the binding assay (see below).

2.2. Mitochondria treatments before binding assays Mitochondria are incubated for 10 min at 4 C in washing buffer complemented with either 50 mM EDTA, or 0.3 mg/ml Proteinase K, or 5 mM Puromycin. Then mitochondria are extensively washed and resuspended in washing buffer supplemented with 1 mM EDTA. Incubation with antibodies (30e35 mg/100 mg mitochondrial

Fig. 1. Naked mRNA is able to bind to isolated mitochondria (A) Standard binding assay: the radioactive transcript (105 cpm) is incubated with purified mitochondria (equivalent of 200 mg of mitochondrial proteins) for 10 min at 25 C. These conditions are used in all binding assays except when other indications are clearly mentioned, i.e. for Fig. 1B and C. After incubation, mitochondria are isolated through a sucrose cushion. RNAs are extracted, separated on formaldehyde agarose gel and transferred onto a membrane. Then the membrane is exposed to detect the radioactive transcript signal. Eventually, the membrane is further hybridized with a mitochondrial 26S rRNA probe and re-exposed. (B) Binding kinetic: GAPDH transcript (105 cpm) is incubated with mitochondria for 5e20 min. The binding signal (transcript) is quantified on Phosphoimager exposure, and the rRNA signal is quantified on the BET-stained gel (rRNA, in inverted colours). The binding signal is then normalized according to the quantity of rRNA, and represented as a function of time. (C) Effect of transcript quantity: different amounts of GAPDH transcript are tested. The binding signal is normalized according to the quantity of rRNA on the gel (rRNA) and represented as a function of transcript amount. (D) Effect of ATP/ADP: GAPDH transcript is incubated with mitochondria in binding buffer (A) or in binding buffer supplemented with 5 mM ATP and 0.04 mM ADP (þA). The binding signal is normalized according to the radioactive 26S rRNA signal (26S). Setting the binding without ATP/ADP (A) to 1.0 standardizes the data. The histogram represents the average data from 7 experiments (n ¼ 7). The error bars correspond to standard errors.



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proteins) is performed for 10 min at 4 C in binding buffer (see below). 2.3. In vitro radioactive transcription GAPDH sequence (coding sequence and 30 UTR from TC147420, http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl? gudb¼potato [9]) and RPL12-p coding sequence (TC182555) have been cloned into pGEM-T easy vector (Promega). In vitro transcription is performed using 2 mg of linearized plasmid, SP6 or T7 RNA polymerase, 40 mCi [32P]-UTP (3000 Ci/mmol) and Riboprobe kit (Promega) according to the manufacter’s instructions. After 2 h at 37 C, transcripts are purified by gel filtration (sephadex G50), phenolic extraction and ethanol precipitation. The pellet is dissolved in H20 at the concentration of 105 cpm/ml (20e25 fmol/ml). 2.4. Binding assays The radioactive transcript (105 cpm) is incubated with isolated mitochondria (equivalent to 200 mg of mitochondrial proteins) for 10 min at 25 C with moderate agitation, in a volume of 50 ml of binding buffer (mannitol 300 mM, KCl 20 mM, DTT 1 mM, malate 1 mM, NADH 1 mM, potassium phosphate 1 mM, MgCl2 5 mM, Hepes-KOH pH 7.2 10 mM, RNasin (Promega) 0.2 ml/50 ml) [17]. After incubation mitochondria are purified through a 27% sucrose cushion [18], washed in 100 ml washing buffer and pelleted. Mitochondria pellets are frozen in liquid nitrogen or directly used for RNA extraction. 2.5. Northern blots

Fig. 2. mRNA binding is independent of ribosomes. EDTA and puromycin are known to release cytosolic ribosomes bound to the mitochondrial surface. The effect of these treatments on mRNA binding is tested. (A and B) Mitochondria are first extracted in washing buffer þ MgCl2 (M), and an aliquot is EDTA-treated (E). Both M and E mitochondria are used for GAPDH transcript binding assay (A), northern blot (B-NB) and western blot (B-WB) analyses. EDTA pre-treatment of mitochondria increases in vitro binding of mRNAs and removes most of cytosolic 5S rRNA bound to mitochondria. However the treatment does not change the level of mitochondrial 5S rRNA or mitochondrial proteins, showing that the integrity of mitochondria is not affected. (C and D) The effect of puromycin is also tested. After EDTA treatment, an aliquot of mitochondria is puromycin treated (P). Both E and P mitochondria are used for GAPDH transcript binding assay (C), northern blot (D-NB) and western blot (D-WB) analyses. This further puromycin treatment has no more effect on mRNA binding, cytosolic 5S rRNA binding and mitochondria integrity. In A and C are shown a representative experiment as well as the average result of 3 and 5 experiments, respectively. Setting the binding with mitochondria prepared in MgCl2 buffer (M, in A) or EDTA-treated mitochondria (E, in C) to 1.0 standardizes the data. In B and D, northern blots (NB) are performed with cytosolic and mitochondrial 5S rRNA probes. Western blots (WB) are performed with antibodies against proteins of the OM (TOM20, VDAC), the IM (NAD9) or the matrix (PDH-E1). Exposition times are different, so the signal intensities cannot be compared between the different panels.

After binding assays, mitochondrial RNAs are extracted using Tri ReagentÒ (Molecular Research) according to the manufacture’s instructions. RNAs are separated by formaldehyde agarose gel electrophoresis and transferred onto HybondNþ (Amersham) membrane. The detection of the radioactive transcript is performed by Phosphorimager exposure and autoradiography of the membrane. The membrane is then hybridized with a 26S rRNA [32P]oligonucleotide probe and re-exposed. The transcript and ribosomal RNA signals are quantified using the Phosphorimager exposures and/or the BET-stained gel image. The transcript signal is normalized with the quantity of rRNA in the sample, to take into account potential variations in RNA extraction. For 5S hybridization, RNAs are separated on 15% (w/v) polyacrylamide/7 M urea gel, electro-transferred onto Hybond-Nþ membrane and hybridized with [32P]-5S oligonucleotide probes [19,20]. The oligonucleotide sequence is 50 -GCCGCCGTTTACCGGGGCTTC CATTC-30 for the mitochondrial 26S rRNA, 50 -GGAGGTCACCCATCCTAGTACTAC-30 for the cytosolic 5S rRNA, and 50 -GGGA TCGGGTGTTTTCACGTC-30 for the mitochondrial 5S rRNA. 2.6. Western blots Fifteen mg of mitochondrial proteins are separated by SDS-PAGE and transferred onto Immobilon-P (Millipore). Western blots are performed as in Ref. [21]. The antibodies are revealed by chemoluminescence using the ECL kit (Amersham Pharmacia Biotech). Antibodies against potato mitochondrial TOM20 and VDAC (@2), Arabidopsis thaliana TOM40, wheat mitochondrial NAD9, and potato VDAC (@1) were kindly provided by H.P. Braun (Hannover University, Hannover, Germany), J. Whelan (University of Western Australia, Perth, Australia), J. M. Grienenberger (Institut de Biologie Moléculaire des Plantes, Strasbourg, France), and A. Dietrich (Institut de Biologie Moléculaire des Plantes, Strasbourg, France),


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Fig. 3. Pre-incubation of mitochondria with TOM complex antibodies strongly affects mRNA binding. Prior to binding assays, mitochondria are incubated with various antibodies (@, in A and B), or are treated with proteinase K (þK, in B). In lane B4, mitochondria are first proteinase K-treated, then incubated with anti-TOM40 antibodies, last used in binding assays. Representative binding assays with GAPDH transcript are shown in A and B. The average results are represented as histograms in C and D. The data are standardized by setting the binding without antibodies (C) or without proteinase K treatment (D) to 1.0. The western blots in (E) show that TOM20 but not all proteins of the OM are affected by



respectively. The antibodies against the maize alpha subunit of PDH-E1 are from GT-Monoclonal-Antibodies. 3. Results 3.1. Naked mRNA is able to bind to isolated mitochondria Among the cytosolic mRNAs found associated to potato mitochondria, GAPDH mRNA is the strongest associated one [9]. Therefore we chose it for in vitro binding assays. In these assays, 105 cpm of a radioactive GAPDH transcript (corresponding to the coding sequence and 30 UTR) are incubated with isolated mitochondria (equivalent of 200 mg of mitochondrial proteins) at 25 C (Fig. 1A). After incubation, mitochondria are purified through sucrose cushion, and mitochondrial RNAs are extracted. Radioactive mRNA is detected in the mitochondrial RNA extract, showing that naked mRNA is able to bind to mitochondria, and that the interaction is strong enough to resist the purification through sucrose cushion. From 5 to 10% of input is bound to mitochondria. The binding is fast and maximum of binding is reached within the 10 first minutes (Fig. 1B). Adding 4 times more transcripts does not saturate the binding (Fig. 1B). The reaction does not need the addition of ATP in the binding buffer (Fig. 1C), suggesting that the process is ATP-independent or that isolated mitochondria can provide enough ATP for the binding. According to these results, we have determined standard conditions for binding assays: transcript (105 cpm) and mitochondria (200 mg of mitochondrial proteins) are incubated in ATP/ADP free binding buffer for 10 min at 25 C (Fig. 1A). 3.2. In vitro mRNA binding is independent of ribosomes Mitochondria are prepared in buffers supplemented with MgCl2, conditions that preserve cytosolic ribosomes at the mitochondrial surface [9]. An aliquot of the preparation is treated with 50 mM EDTA, which removes most of cytosolic ribosomes but does not affect mitochondria integrity (Fig. 2B) [9]. Binding is then performed with both mitochondria preparations. Surprisingly binding is more efficient with EDTA-treated mitochondria (Fig. 2A). In this condition, 10e20% of input is bound to mitochondria. An additional treatment with puromycin, which normally stimulates the release of ribosomes from mitochondria, does not influence anymore neither the binding efficiency nor the quantity of bound cytosolic ribosomes (Figs. 2B and D). These results show that cytosolic ribosomes at the mitochondrial surface do not increase in vitro binding of transcript, but rather suggest that they mask some mRNA binding sites. To favour a more efficient mRNA binding, the next binding assays are performed with EDTA-treated mitochondria. 3.3. Messenger RNA binding is dependent of TOM complex Forty-six proteins have been identified in Arabidopsis thaliana mitochondrial OM [22,23]. Among them, some have been previously identified to be involved in tRNA import in plant mitochondria, such as the Voltage Dependent Anion Channel (VDAC) and components of TOM complex [24,25]. The mitochondrial VDAC was shown to be essential for tRNA translocation through the plant mitochondrial OM, and components of TOM complex are important for tRNA binding at the surface of mitochondria.

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To test the role of such proteins in mRNA binding, mitochondria are pre-incubated with antibodies, and then used in binding assays. As shown in Fig. 3A and C, 2 different VDAC antibodies (@1VDAC and @2VDAC) are used, and both slightly decrease the mRNA binding efficiency (20e25% of loss). By contrast antibodies against components of TOM complex (@TOM20 and @TOM40) induce a drastic inhibition of transcript binding (60e70% of loss) (Fig. 3AeC). As a control, antibodies against the IM protein NAD9 are tested. As expected, these antibodies do not influence the transcript binding to mitochondria (Fig. 3A, C). VDAC proteins are the major proteins of OM and represent more than 50% of their total protein content [26]. They are easily detected in Coomassie-blue stained SDS gel [22] and atomic force microscopy imaging shows their high density in the OM [27]. Moreover we should keep in mind that IgG are very large proteins with an average molecular weight of 160 kDa. Incubation of mitochondria with VDAC antibodies induces a weak loss of binding, suggesting that VDAC can weakly interact with mRNA. However it cannot be excluded that VDACeVDAC antibodies interactions result in an important IgG coating of mitochondria, thus reducing the accessibility to other mRNA binding sites. This would lead to a non-specific reduction of mRNA binding. However such a hypothesis cannot explain the TOM antibodies effects, because TOM components are far less abundant than VDAC and their antibodies effects on mRNA binding are far more efficient (Fig. 3C). These results strongly suggest that subunits of TOM complex or associated components are involved in mRNA binding to mitochondria. 3.4. A component of TOM complex but not TOM20 is essential for binding TOM complex in yeast is composed of receptors (TOM20, TOM22 and TOM70), the import pore (TOM40) and small proteins (TOM5, 6 and7) [4]. In plants, the arrival of plastids has created a selective pressure to maintain the specificity and efficiency of protein import, and the plant mitochondrial protein import apparatus has unique features [4,28]. The unique identified receptor is TOM20, which perform similar functions but does not exhibit sequences similarities with TOM20 from yeast and animals. Other components of plant TOM complex are TOM40 and TOM5, 6, 7 and 9 (Fig. 3F). When mitochondria are proteinase K-treated before incubation with mRNA, a 1.5 increase in mRNA binding is observed (Fig. 3B and D). As TOM20 is completely degraded by proteinase K (Fig. 3E) [29], it cannot be the mRNA receptor. Moreover, the fact that proteinase K treatment induces a more efficient binding suggests that the receptors are now more accessible to mRNAs (Fig. 3F). When using proteinase K-treated mitochondria, the incubation with TOM40 antibodies still induces an inhibition of transcript binding (Fig. 4B lanes 3 and 4, and Fig. 3D), confirming that the mRNA receptor is a component of TOM complex (except TOM20), or a still unknown protein associated with TOM complex. 3.5. The in vitro binding assay does not perfectly reflect the in vivo situation In order to test the specificity of the binding assay, both GAPDH and RPL12-p transcripts are used. In vivo, GAPDH mRNA was found

proteinase K treatment. The effects of mitochondrial treatments on mRNA binding are schematically represented in (F). The TOM complex is shown according to Refs. [23,28]. The numbers 5, 6, 7, 9 refer to the different TOM components accordingly to their size. The question mark represents a potential non-identified component. Pre-treatment of mitochondria with TOM40 antibodies limits the access to mRNA receptors. By contrast proteinase K eliminates TOM20 and increases the accessibility to mRNA receptors. The two treatments have thus antagonist effects.


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4. Discussion

Fig. 4. The RNA binding specificity is partially lost in vitro. Binding assays are performed with 2 different transcripts. One codes for the mitochondrial associated GAPDH protein, the other codes for the plastidial RPL12-p protein. In vivo GAPDH mRNA, but not RPL12-p one, is strongly associated with mitochondria [9]. In vitro, both transcripts are able to bind to mitochondria, but GAPDH binding appears weakly more efficient. 1, 2, 3 correspond to triplicates of the binding assay, that is the incubation of the radioactive transcript (105 cpm) with isolated mitochondria (200 mg of mitochondrial proteins) for 10 min at 25 C. In, input. Only signals in lanes 1 and 2 were taken into account for quantification, because of potential contamination of lane 3 by input.

5 times more associated with mitochondria than RPL12-p transcript [9]. In the in vitro system, GAPDH transcript is found only twice more associated with mitochondria (Fig. 4), suggesting that the binding specificity is partially lost. In the cytosol mRNAs are always associated with cytosolic proteins and/or ribosomes. Some of these factors probably have a role in the specific targeting to mitochondria. By contrast, naked RNAs are used in the in vitro binding assay. These naked RNAs are able to bind to mitochondria showing that a mitochondrial component has affinity for RNA. However the absence of cytosolic factors in the assay could be a reason for the partial loss of specificity.

In this paper, we have developed an in vitro mRNA binding assay with isolated plant mitochondria, in order to identify receptors involved in mRNA docking to the mitochondrial surface. We show that naked mRNAs are able to bind to isolated mitochondria, even if the RNA binding specificity is partially lost. Binding does not need the presence of cytosolic ribosomes at the mitochondria surface, showing that in vitro binding can be translation-independent. We also show that pre-incubation of mitochondria with antibodies raised against VDAC induces a weak reduction of mRNA binding, suggesting that VDAC can weakly interact with mRNA. However we cannot exclude that the effect of VDAC antibodies is non-specific. By contrast pre-incubation of mitochondria with antibodies raised against TOM proteins induces a strong reduction of messenger binding (60e70%), indicating that TOM complex is involved in mRNA binding. The proteinase K pretreatment of mitochondria shows that messenger receptors are insensitive to proteinase K, thus eliminating TOM20 as a potential candidate [23,29]. Mitochondria have to import most of their proteins, and numerous analyses suggest that the corresponding mRNAs are either translated on free polysomes, or are targeted to mitochondria and translated at the mitochondrial surface. Different factors are thought implicated in mRNA targeting to mitochondria. First we show here that mRNA can directly interact with mitochondria. A direct interaction between RNA and plant mitochondria was also shown for tRNA [17,24,25]. Second, cytosolic ribosomes can also bind to the mitochondrial surface. Binding of cytosolic ribosomes to yeast and mammalian isolated mitochondria has been previously demonstrated [30,31]. MacKenzie and Payne have shown that translating ribosomes mainly bind to mitochondria through ribosomal components, but not through the nascent chain [31]. However, up to now, the exact ribosomal component(s) implicated in the interaction are not known. Third, the nascent peptide influences the docking of some mRNAs in yeast. Saint-Georges et al. [12] have shown that translation inhibitors affect the mitochondrial targeting of two third of normally targeted mRNAs and suggest that the other third may be more dependent on direct

Fig. 5. Different factors are expected to be involved in mitochondrial binding of cytosolic mRNAs. The binding of mRNA to mitochondria can be performed through direct mRNA docking (suggested in plant (this work) and yeast [12]), through cytosolic ribosomes associated with mitochondria (shown in yeast and mammals [30,31]; cytosolic ribosomes are also found associated with plant mitochondria [9]), and/or through the nascent peptide (shown in yeast [12,13]). These different mechanisms probably function altogether for the mitochondrial binding of many mRNAs in the different organisms (A). Up to now only a few mitochondrial associated factors have been identified to be involved in mRNA binding to mitochondria. These factors are TOM, Puf3p and VDAC (A, B, C). TOM complex seems implicated in mRNA binding by a direct docking of mRNA and through the interaction of the nascent peptide [13,14,32], this work) (A). Puf3p, a RNA binding protein interacting with Mdm12p, is essential for mitochondrial targeting of some mRNAs in yeast, and eventually works with TOM complex [12,13] (B). Last, the role of VDAC as an mRNA receptor in plants is questioned (this work) (C). To simplify the figure, the interactions with ribosomes or TOM complex, although probably occurring in vivo, are not shown in the representation of mRNA binding to Puf3p and VDAC (B and C). For example, it is known that cytosolic ribosomes are associated with mitochondria in numerous organisms [5,9], and the interactions between Puf3p and TOM have been demonstrated in yeast [13]. Moreover other not yet identified mitochondrial factors are probably involved in mRNA docking in the different organisms.



mRNAemitochondria interactions. In accordance with these results, mitochondrial targeting of ACO1 mRNA in yeast appears linked to translation and to the presence of a functional MTS [13]. By contrast the mutation of the initiation codon has a poor effect of OXA1 mRNA localization [14]. In summary the association of mRNA with mitochondria could be performed through direct mRNA docking, through cytosolic ribosomes bound to mitochondria, or through the nascent peptide (Fig. 5A). These 3 possibilities are not exclusive, but rather overlap. The respective role of each interaction seems to change from one mRNA to the other and need further investigations. The next question is the identification of mitochondrial receptors. TOM complex has been already shown to be involved in mRNA binding. Among the mRNA coding for mitochondrial proteins, more than 80% were mislocalized in a tom20 deletion yeast strain [13], and Eliyahu et al. have demonstrated a role of TOM20 in MTS-dependent mRNA localization. Gadir et al. [14] have also shown that mitochondrial association of some mRNAs is affected in different tom mutants in yeast (tom6, 7, 20, 70). However this effect is not obligatory linked to translation. For example OXA1 mRNA targeting to mitochondria depends on TOM complex but is not strongly affected by initiation codon mutation or MTS deletion [14,32]. Our results in this paper suggest that naked mRNA can bind to plant mitochondria through TOM complex. TOM complex was also shown implicated in tRNA binding to the mitochondrial surface in plants [24]. All together, these results suggest that TOM complex is implicated in mRNA binding to the mitochondrial surface by at least 2 mechanisms which are not exclusive, through the interaction of the nascent peptide, and by a direct docking of mRNA (Fig. 5A). Some mRNAs are also targeted to mitochondria without the need of TOM complex, suggesting alternatively receptors [13]. A RNA binding protein, Puf3p, has been identified as essential for mitochondrial targeting of a subset of mRNAs in yeast [12]. Puf3p is known for its role in mRNA decay, and is also a peripheral protein associated with the cytosolic face of OM [33]. Puf3p was identified as interacting with Mdm12p, a component of ERMES complex in the yeast OM [33e35]. These results suggest another way for mRNA binding to the mitochondrial surface (Fig. 5B). However it should be noted that many mitochondrial-targeted mRNAs that depend on Puf3p also depend on TOM complex, showing interactions between Puf3p and TOM functions [13]. Last, VDAC has been shown to have affinity with plant tRNAs [24], and could also weakly interact with mRNAs (this work) (Fig. 5C). Altogether these results show the incredible complexity of mitochondrial targeting processes and suggest that there is not one unique pathway, but rather different overlapping routes for the intracellular localization of mRNAs to the mitochondrial surface, which emerges as an essential process in mitochondria biogenesis.

Acknowledgements We thank Laura Peter and Elodie Ubrig for technical help. This work was supported by Université de Strasbourg and Centre National de la Recherche Scientifique (CNRS), by the French Agence National de la Recherche (ANR-09-BLAN-0240-01) and by the French National Program “Investissement d’Avenir” (Labex MitoCross). MM has a fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche.

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Cytosolic and mitochondrial surface factor-independent import of a synthetic peptide into mitochondria.

Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA.

Evidence That Antibiotics Bind to Human Mitochondrial Ribosomal RNA Has Implications for Aminoglycoside Toxicity.

Targeting of a cytosolic protein to the nuclear periphery.

Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

Cytosolic PINK1 escapes from mitochondria to promote dendritic outgrowth.

Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae.

Commentary: Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage-Dependent Anion Channel 1 (VDAC1) in Saccharomyces cerevisiae Mitochondria.

Targeting of the master receptor MOM19 to mitochondria.

Targeting mitochondria to restore failed adaptation to exercise in diabetes.

Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal.

Capacity of ribosomal RNA promoters of E. coli to bind RNA polymerase.

Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli.

Targeting of hexokinase 1 to liver and hepatoma mitochondria.

Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting.

Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria.

Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors.

Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

Targeting molecular hydrogen to mitochondria: barriers and gateways.

OM14 is a mitochondrial receptor for cytosolic ribosomes that supports co-translational import into mitochondria.

Bioprotective carnitinoids: lipoic acid, butyrate, and mitochondria-targeting to treat radiation injury: mitochondrial drugs come of age.

Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I.

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?