Bovine TWINKLE and mitochondrial ribosomal protein L43 genes are regulated by an evolutionary conserved bidirectional promoter

Abstract

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 transcription 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.

Keywords: ATP; AcGFP1; Aequorea coerulescens green fluorescent protein 1; Bidirectional promoter; C. elegans; Caenorhabditis elegans; DMEM; DNA; Discosoma striata Red; DsRed; Dulbecco's Modified Eagle's Medium; E. coli; EDTA; Escherichia coli; FBS; MRPL43; MTDPS7; Mitochondrial DNA; Mya; OMIM; PBS; PCR; POEA3; RNA; Ribosomal protein L43; TFBS; TWINKLE; adenosine triphosphate; base pair; bp; deoxyribonucleic acid; ethylenediaminetetraacetic acid; foetal bovine serum; million years ago; mitochondrial DNA; mitochondrial DNA depletion syndrome-7; mitochondrial ribosomal protein L43; mtDNA; online Mendelian inheritance in man; phosphate buffered saline; polymerase chain reaction; progressive external ophtalmoplegia 3; ribonucleic acid; transcription factor binding site.