Modulation of Mrps12/ Sarsm promoter activity in response to mitochondrial stress

Abstract

Transcription from the bidirectional promoter of two mouse genes encoding components of the mitochondrial translational apparatus, mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm), was shown previously to be dependent upon an array of four CCAAT boxes, interacting with the transcription factor NF-Y. Here we report that the homologous human promoter is governed by a CCAAT box array acting in an essentially similar manner. Analysis of the transcriptional response of both the human and mouse promoters to various mitochondrially acting toxins, including inhibitors of mitochondrial protein synthesis, and agents that bring about uncoupling or respiratory chain inhibition, produced either of two distinct outcomes, depending on the cell type and the conditions used. In mouse C2C12 myoblasts, human HEK293 cells or U2OS osteosarcoma cells, plus HeLa cells at high drug doses or mouse 3T3 fibroblasts subjected to prolonged drug exposure, a dose-dependent, bidirectional suppression of transcription was observed. In 3T3 cells subjected only to pre-treatment with the drugs, bidirectional Mrps12/ Sarsm promoter activity was strongly stimulated. A similar, though weaker stimulation was observed at lower drug doses in HeLa cells. Reporter studies using mutated variants of the mouse promoter construct indicated that the stimulation of promoter activity in response to mitochondrial OXPHOS stress in 3T3 cells was independent of the CCAAT box array and of putative binding sites for NRF-2, AP-1 and other transcription factors, whereas transcriptional downregulation under prolonged mitochondrial stress was CCAAT box-dependent. Promoter stimulation was correlated with mitochondrial ROS production, which may be a crucial component in its signalling.