Short Communication Bovine TWINKLE and mitochondrial ribosomal protein L43 genes are regulated by an evolutionary conserved bidirectional promoter Cédric Meersseman a,b,c,d , Véronique Léjard a,b , Emmanuelle Rebours a,b , Mekki Boussaha a,b , Abderrahman Maftah c,d , Daniel Petit c,d , Dominique Rocha a,b, ⁎ a INRA, UMR1313, Unité Génétique Animale et Biologie Intégrative, Domaine de Vilvert, F-78352 Jouy-en-Josas, France b AgroParisTech, UMR1313, Unité Génétique Animale et Biologie Intégrative, Domaine de Vilvert, F-78352 Jouy-en-Josas, France c INRA, UMR1061 Génétique Moléculaire Animale, F-87060 Limoges, France d Université de Limoges, UMR1061 Génétique Moléculaire Animale, F-87060 Limoges, France abstract article info Article history: Accepted 30 November 2013 Available online 19 December 2013 Keywords: TWINKLE Mitochondrial DNA Ribosomal protein L43 Bidirectional promoter TWINKLE is a mitochondrial DNA helicase playing an important role in mitochondrial DNA replication. In human, mutations in this gene cause progressive external ophtalmoplegia and mitochondrial DNA depletion syndrome-7. TWINKLE is well conserved among multicellular eukaryotes and is believed to be a key regulator of mitochondrial DNA copy number in mammals. Despite its involvement in several diseases and its important function in mitochondrial DNA metabolism, nothing is known about the regulation of the expression of TWINKLE. We have analysed the 5′-flanking genomic region of the bovine TWINKLE gene and found it was localised adjacent to the MRPL43 gene in a head-to-head orientation, suggesting that both genes are regulated by a shared bidirectional promoter. The bovine 75-bp long intergenic region shows substantial homology across different species and contains several conserved putative transcrip- tion factor binding sites. A TATA box, however, was lacking. Using a dual fluorescent reporter system and transient transfection assays, we have analysed the bovine intergenic region between TWINKLE and MRPL43. This small genomic fragment showed a bidirectional promoter activity. As the TWINKLE/MRPL43 bidirectional promoter tested was highly conserved, it is likely that the results we obtained here in cattle may be extended to the other species. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The TWINKLE or chromosome 10 open reading frame 2 (C10ORF2) gene was originally identified in a search for mutations associated with chromosome 10q24-linked autosomal dominant progressive external ophthalmoplegia (POEA3, OMIM 609286), which is a human disorder with exercise intolerance, muscle weakness, peripheral neu- ropathy, deafness, ataxia, cataracts and hypogonadism. The protein encoded by TWINKLE/C10ORF2 has a mitochondrial targeting sequence at its N-terminus. Homology searches revealed sequence similarities between this protein and the bacteriophage T7 gene 4 protein, which contains both the DNA helicase and the primase activities needed at the bacteriophage replication fork. The TWINKLE protein colocalises with mitochondrial DNA (mtDNA) in nucleoprotein complexes desig- nated mitochondrial nucleoids, and its name derives from the unusual punctate, star-like staining reminiscent of twinkling stars (Spelbrink et al., 2001). The TWINKLE gene product belongs to a class of hexameric helicases, and its amino acid sequence is well conserved among multi- cellular eukaryotes (Shutt and Gray, 2006). TWINKLE functions as an ATP-dependent DNA helicase and unwinds short stretches of double-stranded DNA in the 5′ to 3′ direction, stimu- lated by the mitochondrial single-stranded DNA-binding protein (Farge et al., 2008; Korhonen et al., 2003; Sen et al., 2012). Along with mito- chondrial single-stranded DNA binding protein and mtDNA polymerase gamma, it is believed that TWINKLE forms a processive replication ma- chinery, which can use double-stranded DNA as a template to synthetise single-stranded molecules (Korhonen et al., 2004). TWINKLE function is thought to be critical for maintenance of mtDNA integrity as TWINKLE seems to play an important role in mtDNA replication. To elucidate the in vivo role of TWINKLE in mtDNA maintenance, Tyynismaa and collaborators generated two transgenic mouse lines overexpressing wild-type Twinkle (Tyynismaa et al., 2004). They could Gene 537 (2014) 154–163 Abbreviations: AcGFP1, Aequorea coerulescens green fluorescent protein 1; ATP, aden- osine triphosphate; bp, base pair; C. elegans, Caenorhabditis elegans; DMEM, Dulbecco's Modified Eagle's Medium; DNA, deoxyribonucleic acid; DsRed, Discosoma striata Red; E. coli, Escherichia coli; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine serum; MRPL43, mitochondrial ribosomal protein L43; mtDNA, mitochondrial DNA; MTDPS7, mi- tochondrial DNA depletion syndrome-7; Mya, million years ago; OMIM, online Mendelian inheritance in man; PBS, phosphate buffered saline; PCR, polymerase chain reaction; POEA3, progressive external ophtalmoplegia 3; RNA, ribonucleic acid; TFBS, transcription factor binding site. ⁎ Corresponding author at: UMR1313, Unité de Génétique Animale et Biologie Intégrative, Centre INRA de Jouy-en-Josas, Domaine de Vilvert, F-78352 Jouy-en-Josas cedex, France. Tel.: +33 1 34 65 24 22; fax: +33 1 34 65 24 78. E-mail address: dominique.rocha@jouy.inra.fr (D. Rocha). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.088 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene