Abstract
Editor’s summaryThe secondary structure of mRNA plays crucial roles in gene expression. Moran et al. developed a chemical labeling approach (mitoDMS-MaPseq) and used a clustering algorithm to reveal the folding patterns of mitochondrial mRNA (mt-mRNA) in wild-type and LRPPRC-deficient cells. LRPPRC is a protein crucial for mt-mRNA maintenance and translation and acts as an mRNA holdase, influencing mRNA folding. Analyses of genome-wide and mt-mRNA–specific folding features revealed mRNA-programmed translational pausing and programmed ribosomal frameshifting. These findings establish a pivotal layer in mitochondrial gene expression, providing mt-mRNA folding maps for diverse physiological and pathological studies. —Di JiangINTRODUCTIONExpression of the human mitochondrial genome (mtDNA) provides 13 essential protein subunits of the oxidative phosphorylation (OXPHOS) enzymatic complexes that catalyze aerobic energy transduction and support life. The mtDNA is a double-stranded 16.569-Kb molecule with heavy (H) and complementary light (L) strands. Transcription spans almost their entire length, producing two long polycistronic transcripts that are processed to yield 11 mature mRNAs, as two transcription units remain unprocessed as bicistronic elements containing overlapping and –1-shifted open reading frames (ORFs) encoding ATP8 and ATP6 or ND4L and ND4. The mtDNA also encodes the two ribosomal RNAs (12S and 16S rRNA) and 22 transfer RNAs required for synthesizing these proteins in mitochondrial ribosomes. Over the past five decades, the molecular machineries and mechanisms governing mtDNA transcription and mitochondrial messenger RNA (mt-mRNA) stability, processing, modification, and translation have been progressively characterized and remain the subject of intense investigations. However, our knowledge of the mt-mRNA folding landscape or structurome is very limited, which has hindered our mechanistic understanding of mtDNA gene expression and its regulation.RATIONALEObtaining mRNA structure data within intact mitochondria is critical for understanding its biological context. To this goal, we have adapted an approach for the chemical probing of mRNA structures using dimethyl sulfate (DMS), which methylates the base-pairing faces of adenines and cytosines that are unpaired and accessible. Mitochondrial DMS mutational profiling with sequencing (mitoDMS-MaPseq) is a high-throughput, genome-wide RNA structure probing strategy that takes advantage of a high-fidelity and processive thermostable group II reverse transcriptase (TGIRT) enzyme that converts DMS modifications in the RNA to mismatches in the complementary DNA. DMS reactivity information is then used as a constraint in RNA folding algorithms based on thermodynamics to obtain highly accurate secondary structure models.RESULTSHaving established a reproducible and accurate mitoDMS-MaPseq approach, we have investigated the structure of mt-mRNAs within functional human mitochondria. We used mitochondria isolated from wild-type cells and cells lacking the leucine-rich pentatricopeptide repeat–containing protein (LRPPRC), a pivotal regulator of mt-mRNA stability, polyadenylation, and translation. Our comparative analysis extended to in vitro synthesized and folded mt-mRNAs. Our findings elucidate LRPPRC’s role as a holdase that contributes to maintaining mt-mRNA folding and efficient translation. The mt-mRNA structural insights in wild-type mitochondria and metabolic labeling have unveiled potential mechanisms of gene expression. They include mRNA-programmed translational pausing to support the synthesis of COX1, a hydrophobic protein with multiple transmembrane domains, and a specific case of mRNA-programmed ribosome frameshifting (PRF) followed by termination-reinitiation events to coordinate translation of the two ORFs in the bicistronic ATP8/6 transcript. Furthermore, by using the clustering algorithm DREEM (detection of RNA folding ensembles using expectation-maximization), we have identified coexisting alternative conformations adopted by each transcript, capturing the dynamic nature of mt-mRNA folding in structural ensembles.CONCLUSIONOur findings define the mt-mRNA folding landscape, offering insight into posttranscriptional regulation of mitochondrial gene expression. Additionally, we present a strategy that enables the investigation of mt-mRNA folding across cells and tissues and their role in regulating mitochondrial gene expression during development, diseases, and aging.