Abstract
Messenger RNAs (mRNAs) fold to adopt secondary and tertiary structures that often have essential regulatory functions in their synthesis, processing, translation, and turnover. In mammalian cells, mitochondria contain a genome that encodes for 13 protein components of the oxidative phosphorylation system that drives aerobic energy transduction. These 13 proteins are translated from 9 monocistronic mRNAs and 2 bicistronic transcripts with overlapping reading frames. The native structures of mitochondrial mRNAs (mt-mRNAs) have not been investigated, and therefore fundamental molecular determinants of mitochondrial gene expression remain unknown. Here, we have optimized and applied a mitochondrial dimethyl sulfate (DMS) probing technique coupled with mutational profiling through next-generation sequencing (mitoDMS-MaPseq) to assess the native architecture of all mitochondrial mRNAs. The data provide insights into mitochondrial mRNA biology and, in combination with biochemical data, support the existence of translation regulatory mechanisms. The approach allows for exploration of interactions of mRNAs with ligands such as proteins and associated conformational and functional changes. We have focused on the pediatric neurodegeneration protein LRPPRC, required for mRNA stability, polyadenylation, and delivery to the mitoribosome for translation of most mRNAs. In the absence of LRPPRC, mRNAs are degraded, but those that remain stable fold differently than in wild-type cells. In summary, the mt-mRNA folding landscape provides a key layer of post-transcriptional regulation of mitochondrial gene expression. The approach described here will allow elucidation of mt-mRNA structuromes in any cell type or tissue and help illuminate novel roles of mRNA secondary structure in mitochondrial gene expression regulation, in development, diseases, and the aging process.