Codon optimality modulates protein output by tuning translation initiation.

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

OXPHOS deficiencies affect peroxisome proliferation by downregulating genes controlled by the SNF1 signaling pathway.

How environmental cues influence peroxisome proliferation, particularly through organelles, remains largely unknown. Yeast peroxisomes metabolize fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, produced by these pathways enter mitochondria for ATP production and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH-shuttling- and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by a high AMP/ATP ratio. In OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Δgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2, and NRG1) controlled by SNF1 signaling. Our results are interpreted in terms of a mechanism by which peroxisomal and mitochondrial proteins and/or metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol, and the nucleus. We discuss the physiological relevance of this work in the context of human OXPHOS deficiencies.