UC San Diego

Mitochondria are organelles whose function, protein composition, size, and morphology are highly variable and regulated in response to nutrient availability and other environmental conditions. Their crucial role in metabolism involves the production of adenosine triphosphate (ATP), the energy currency of the cell, by the oxidative phosphorylation pathway. TEM studies observed ribosomes enriched at the mitochondrial surface, suggesting that mRNAs localize co-translationally. mRNA localization is a post transcriptional method for regulating gene expression in parallel with transcriptional methods. While mRNA localization is a way to control protein production or limit translation activity to specific cellular locations, the potential of mRNA localization as a strategy for altering the composition of mitochondrial proteins in different environmental conditions has not been explored. For brewer’s yeast, 99% of mitochondrial proteins are encoded in the nuclear genome. To ensure mitochondrial function, nuclear-encoded proteins are imported into the mitochondria through mitochondrial translocases on the outer mitochondrial membrane. mRNA localization is implicated in mitochondrial protein homeostasis along two axes: it helps synchronize the nuclear and mitochondrial genome translation programs to ensure proper stoichiometry of nuclear- and mitochondrial-encoded proteins and it is required for initiating the co-translational import of highly hydrophobic nascent peptides that are vulnerable to aggregation and misfolding in the cytosol. While it is clear that mRNA localization is important for mitochondrial biogenesis and homeostasis, the mechanism of localization has not been fully elucidated for mRNAs that do not have known RNA-binding proteins partners that regulate their localization. More than 200 genes associate to the mitochondria after translating a 5’ amphiphilic mitochondria targeting sequence (MTS) that can interact with translocation machinery only after the mRNA-ribosome complex has found the mitochondria through diffusive search. Additionally, the nascent peptides produced by co-translationally localized mRNAs bind to the chaperones Hsp70 (in brewer’s yeast) and Hsp90 (in mammalian cells) that are implicated in the proper recognition of the preprotein by other components of translocation machinery. Based on our understanding of the MTS-driven mechanism of mRNA localization, I developed a mathematical model and stochastic simulation of translation, peptide signal maturation, and mRNA diffusion in cells of varying mitochondrial volume. After reproducing experimental observations of mRNA localization in diverse cellular states, including in fermentative versus respiratory metabolic conditions, we predicted that increasing translation duration would drive mRNA localization in vivo. However, quantitative microscopy in brewer’s yeast reveals that ribosome stalls downstream of the MTS drive localization regardless of translation duration. This has given us new insights into the peptide signal maturation component of MTS-driven localization mechanism, and has been incorporated into the stochastic simulation as well as experimental design. The mechanism of mRNA localization is based on fundamental processes like translation and diffusion instead of relying on gene-specific or condition-specific regulatory factors. Therefore, we postulate that it is conserved to mammalian systems given the high degree of conservation of mammalian genes and peptide-binding chaperones. We find that our stochastic simulation of translation, peptide signal maturation, and mRNA diffusion can reproduce experimental observations of mRNA localization behaviors in brewer’s yeast and mammalian cells, indicating that the biophysical mechanism of MTS-driven localization is conserved between eukaryotes. The combination of translation and diffusion kinetics is a novel mechanism for regulating mitochondrial gene expression post-transcriptionally across eukaryotes and adds to our understanding of mitochondrial homeostasis and mitochondrial biogenesis in shifting environmental conditions.