The human mitochondrial mRNA structurome reveals mechanisms of gene expression

The mammalian mitochondrial genome encodes thirteen proteins of the oxidative phosphorylation system, crucial in aerobic energy transduction. These proteins are translated from 9 monocistronic and 2 bicistronic transcripts, whose native structures remain unexplored, leaving fundamental molecular determinants of mitochondrial gene expression unknown. To address this knowledge gap, we developed a mitoDMS-MaPseq approach and used DREEM clustering to resolve the native mt-mRNA structurome in human mitochondria. In this way, we gained insights into mt-mRNA biology and translation regulatory mechanisms, including a unique translational frameshifting for the ATP8/ATP6 transcript. Furthermore, absence of the mt-mRNA maintenance factor LRPPRC led to a mitochondrial transcriptome structured differently, with specific mRNA regions exhibiting increased or decreased structuredness. This highlights the role of LRPPRC in maintaining mRNA folding to promote mt-mRNA stabilization and efficient translation. In conclusion, our mt-mRNA folding maps reveal novel mitochondrial gene expression mechanisms, serving as a detailed reference and tool for studying them in different physiological and pathological contexts. Overall design: To investigate the structure of human mitochondrial mRNAs. Briefly, mitochondria isolated from HEK-293T cells were incubated with DMS and RNA was extracted from isolated mitochondria. An off the shelf IDT RNAseq library preparation kit was used in conjunction with TGIRT to mediated mutagenesis where DMS modifications occur. This sequencing data was then aligned to the mitochondrial genome and structural models were developed using Detection-of-RNA-folding-Ensembles-using-Expectation-Maximization (DREEM, Rouskin Lab) and RNAstructure (Mathews lab).