Regulation of mitochondrial transcription during anoxia-induced quiescence in the brine shrimp Artemia franciscana

Mitochondria isolated from gastrula-stage embryos of the brine shrimp Artemia franciscana were used to study mechanisms of transcriptional regulation during anoxia-induced quiescence. In response to anoxia, embryos undergo a rapid, reversible, and profound downregulation of metabolism and development characterized by a sharp drop in intracellular pH (pH_i) and ATP concentrations. A variety of aspects of transcription were studied using isolated mitochondria to elucidate mechanisms governing RNA synthesis and degradation under conditions promoting anoxia-induced quiescence. Transcription rate at pH 7.9 was reduced by 80% when pH was lowered to 6.3, which mimics the in vivo change seen in embryos under anoxia. Although transcription did not depend on oxidative phosphorylation when external ATP was available, ATP inhibition at subsaturating UTP levels was relieved when UTP was at saturating, physiological levels.; Levels of four selected mitochondrial mRNAs were measured under conditions of low (pH 6.4) and high pH (pH 7.9) with or without oxygen, and parameters of mRNA half-life and degradation were calculated. Half-lives were significantly increased under both anoxia and low pH relative to controls (normoxia, pH 7.9). Under the same conditions, the degree of polyadenylation in each of the mRNAs was measured and correlated with message stability. In contrast to the eukaryotic nucleus, polyadenylation was associated with decreased message stability.; Transcription initiation during in vitro incubations contributed substantially to measured nucleotide incorporation in vitro under control conditions (about 78% at pH 7.8), but at pH 6.4, the contribution was reduced to 32% of transcription under either anoxia or aerobic conditions. In organello footprinting showed that protein dissociation from mtDNA at pH 6.4 is unlikely to be a mechanism responsible for decreased initiation, since no effect of in organello treatment was seen on footprinting patterns. However, these patterns did not change with in organello treatment. Under conditions of anoxia-induced quiescence, mitochondrial transcription decreased by 77% relative to normoxic controls. Acute effects of anoxia in vitro were also measured at pH 7.9 (39% decrease) and at pH 6.4 (70%). Important roles for both oxygen removal and pH_i acidification have been shown in the downregulation of mitochondrial transcription during anoxia-induced quiescence.