Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle

Mitochondria play a critical role in cellular metabolism primarily through hosting the oxidative phosphorylation (OXPHOS) machinery that is encoded by mitochondrial DNA (mtDNA) and nuclear DNA, with each gene expression system being regulated by a distinct set of mechanisms. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to gain insights into the principles underlying the regulation of mtDNA-encoded protein abundance. Our analysis revealed a unique balance between the rapid turnover and high accumulation of mt-mRNA, leading to a 700-fold higher transcriptional output compared to nuclear-encoded OXPHOS genes. Additionally, we observed that mt-mRNA processing and its association to the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. These data resulted in a model of mtDNA expression that is predictive across cell lines, revealing that differential turnover and translation efficiencies are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome,  French–Canadian type, we found that the responsible nuclear-encoded gene, LRPPRC, disrupts OXPHOS biogenesis predominantly through altering mt-mRNA stability.  Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle for decoding the regulatory principles of mtDNA expression. TimeLapse-seq and TimeLapse-seq-mitoribosome IP data for HeLa S3, HEK293T, and LRPPRC KO (HEK293T) cells using both 50 uM and 500 uM 4sU. Also includes HeLa RNA-seq, mitochondrial polysome RNA-seq, and K562 cytosolic and mitochondrial ribosome profiling