A kinetic dichotomy between mitochondrial and nuclear gene expression drives OXPHOS biogenesis

Abstract

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 genome separately regulated in their respective compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. Here, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to gain insight into which steps of gene expression experience the most regulatory control. Our analysis revealed a unique balance between the rapid turnover and high accumulation of mt-mRNA, leading to a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. Additionally, we observed that mt-mRNA processing and its association with 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 human 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 disrupting the responsible nuclear-encoded gene, LRPPRC, perturbs 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.

The complexity and dynamics ofin organellotranslation assessed by high-resolution mitochondrial ribosome profiling

“…We further measured mRNA half-lives by 5′-bromo-uridine immunoprecipitation chase–deep sequencing (BRIC-Seq) 26,52,53 and found a similar range of mRNA half-lives to those previously reported 54,55 (Figure S2B), although the numbers for individual mRNAs differed between our study and those studies (Table S2). This measurement allowed us to calculate the lifetime translation cycles before transcript decay; we determined that the average number of mitochondrial mRNAs used for protein synthesis was 31.8 (Figure S2C).…”

“…These inefficiencies may be compensated for by the high expression of mitochondrial mRNAs (Figure S10O). A recent study by Churchman’s group 55,118 reached a similar conclusion regarding a slow translation rate in mitochondria. Ultimately, the protein supply from the mitochondrial matrix and the cytosol should be balanced for the maintenance of proper proteostasis 16,119,120 .…”

Mitochondrial protein synthesis quality control

“…Evidence points to the synthesis of incomplete mRNA transcripts that are subsequently polyadenylated [ 13 , 14 ]. Importantly, there appears to be no surveillance system to prevent translation initiation on mitochondrial mRNAs with aberrant 3′ ends [ 14 , 15 ]. Thus, translation of these partial polyadenylated mRNAs would generate fusion open reading frames (ORFs) whose nascent chains would misfold during synthesis or in the inner membrane, impinging on the proteostasis of the organelle.…”

“…Elegant research has reconstituted the processing events mediated by the RNase P complex composed of TRMT10C, HSD17B10, and PRORP on single tRNA substrates [ 17 , 19 , 20 ]. Recent experimental evidence, however, suggests that low level errors in the 5’ tRNA processing arise that would in turn affect the 3′ end of six mitochondrial mRNAs [ 14 , 15 ]. Deep sequencing of translating mitochondrial ribosomes revealed that these transcripts consist of the mRNA with a fragment of the 3′ flanking tRNA sequence in frame ( Fig.…”

“…Since there appears to be no surveillance mechanism to prevent translation of mRNAs with aberrant 3′ ends [ 14 , 15 ], understanding how defects in translation elongation arise, are recognized, and resolved is critical for developing an integrated view of mitochondrial protein synthesis quality control. The mitochondrial ribosome provides an ideal hub to coordinate these events.…”