TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane Sylvain Hanein a, ⁎, Mathilde Garcia b , Lucas Fares-Taie a , Valérie Serre a, c , Yves De Keyzer a , Thierry Delaveau b , Isabelle Perrault a , Nathalie Delphin a , Sylvie Gerber a , Alain Schmitt d, e, f , Jean-Marc Masse d, e, f , Arnold Munnich a , Josseline Kaplan a , Frédéric Devaux b , Jean-Michel Rozet a, ⁎ a Institut National de la Santé et de la Recherche Médicale (INSERM) U781, Départment de Génétique, Fondation Imagine, Université Paris Descartes-Sorbonne Paris Cité, 75015 Paris, France b Génomique des microorganismes, UMR 7238 CNRS-Université Pierre et Marie Curie, 75006 Paris, France c Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France d INSERM, U1016, Institut Cochin, 75014 Paris, France e CNRS, UMR8104, 75014 Paris, France f Université Paris Descartes, Sorbonne Paris Cité, 75015 Paris, France abstract article info Article history: Received 3 January 2013 Received in revised form 13 February 2013 Accepted 26 February 2013 Available online 13 March 2013 Keywords: TMEM126A Optic neuropathy Mitochondrial inner membrane Cristae Mitochondria-localized mRNA MLR Background: Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently iden- tified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cel- lular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function. Methods: A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluo- rescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out. Results: TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochon- drial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization. Conclusions: TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON. General significance: Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins. © 2013 Elsevier B.V. All rights reserved. Biochimica et Biophysica Acta 1830 (2013) 3719–3733 Abbreviations: HON, hereditary optic neuropathy; RGC, retinal ganglion cell; mtDNA, mitochondrial DNA; LHON, Leber's Hereditary Optic Neuropathy; OPA, optic atrophy; TMEM126A, transmembrane protein 126A; IMS, intermembrane space; OM, outer membrane; IM, inner membrane; SAPS, Statistical Analysis of Protein Sequences; TMEM126B, transmembrane protein 126B; DUF, domain of unknown function; MLR, mitochondrial-located mRNA; AA, amino acid; FISH, fluorescent in situ hybridization; UQCRC1, ubiquinol-cytochrome c reductase core protein I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RNA12S, mitochondrially encoded 12S RNA; mt-rRNA, mitochondrial ribo- somal RNA; DPBS, Phosphate Buffered Saline; MB, mitochondrial buffer; MAPK1, mitogen-activated protein kinase 1; MAPK3, mitogen-activated protein kinase 3; MT-CO2, mt-DNA encoded cytochrome c oxidase II; NDUFA9, NADH dehydrogenase ubiquinone 1 alpha subcomplex 9 39 kDa; VDAC1, voltage-dependent anion channel 1; CYCS, cytochrome c so- matic; NDUFB6, NADH dehydrogenase ubiquinone 1 beta subcomplex 6 17 kDa; PDHA1, pyruvate dehydrogenase lipoamide alpha 1; BCL2, B-cell CLL/lymphoma 2; ATP5B, ATP synthase H + transporting, mitochondrial F1 complex beta polypeptide; SDHA, succinate dehydrogenase complex subunit A flavoprotein; TEM, transmission electron microscopy; qFISH, quantitative fluorescent in situ hybridization; MTS, mitochondrial targeting leader sequence; TM, transmembrane; Puf3p, Pumilio-Fem-3-binding factor (FBF) (Puf) RNA binding protein; UTR, untranslated region; PUM1, pumilio protein 1; PUM2, pumilio protein 2; miRNA, micro RNA; CDS, coding sequence; G3BP1, GTPase activating protein (SH3 domain) binding protein 1; YB-1, Y box binding protein 1 ⁎ Corresponding authors at: Institut National de la Santé et de la Recherche Médicale (INSERM) U781, Départment de Génétique, Hôpital des Enfants Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. Tel.: +33 144 49 51 56; fax: +33 147 83 32 06. E-mail addresses: sylvainhanein@hotmail.com (S. Hanein), jean-michel.rozet@inserm.fr (J.-M. Rozet). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.02.025 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen