Summary Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance rate of translation elongation, whereas non-optimal codons slow elognatioon. Codon usage also controls ribosome traffic on mRNA. These conclusions were further supported by ribosome profiling results in vitro and in vivo with template mRNAs designed to increase signal to noise. Finally, we demonstrate that codon usage regulates protein function by affecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding.

Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms.

Although lanthanide-doped upconversion nanoparticles (UCNPs) have shown great promise in biosensing and bioimaging owing to their excellent photophysical properties, researchers are facing a bottleneck of upconversion (UC) probes which is the limited signal-to-background ratio (SBR). Since UC nanoprobes are basically constructed with a luminescence resonance energy transfer (LRET) process to provide "off−on" signals, the SBR level is principally decided by the luminescence quenching efficiency which is very difficult to further improve through existing approaches. Herein, we put forward a new strategy for fabricating UC nanoprobes using an organic dye as target-modulated sensitizing switch. The dye functions as both the recognition unit for target and a potential sensitizer for upconversion luminescence (UCL). The reaction of the dye with target modulates its photophysical properties, which switches on the sensitization and affords a significantly improved SBR. The idea is validated with a proof-of-concept UC nanoprobe for glutathione (GSH) detection with the SBR of ∼30 (versus a SBR of less than 10 for most current UC nanoprobes). This probe showed good performance in GSH sensing both in vitro and in vivo. Our results indicate that the target-modulated sensitization is a useful new strategy to build UC nanoprobes. And we can reasonably expect that the breakthrough of SBR limit will make UC nanoprobe a more powerful tool in future studies.

Codon usage bias, the preference for certain synonymous codons, is found in all genomes. Although synonymous mutations were previously thought to be silent, a large body of evidence has demonstrated that codon usage can play major roles in determining gene expression levels and protein structures. Codon usage influences translation elongation speed and regulates translation efficiency and accuracy. Adaptation of codon usage to tRNA expression determines the proteome landscape. In addition, codon usage biases result in nonuniform ribosome decoding rates on mRNAs, which in turn influence the cotranslational protein folding process that is critical for protein function in diverse biological processes. Conserved genome-wide correlations have also been found between codon usage and protein structures. Furthermore, codon usage is a major determinant of mRNA levels through translation-dependent effects on mRNA decay and translation-independent effects on transcriptional and posttranscriptional processes. Here, we discuss the multifaceted roles and mechanisms of codon usage in different gene regulatory processes. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.

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Codon usage bias is a fundamental feature of all genomes and plays an important role in determining gene expression levels. The codon usage was thought to influence gene expression mainly due to its impact on translation. Recently, however, codon usage was shown to affect transcription of fungal and mammalian genes, indicating the existence of a gene regulatory phenomenon with unknown mechanism. In Neurospora, codon usage biases strongly correlate with mRNA levels genome-wide, and here we show that the correlation between codon usage and RNA levels is maintained in the nucleus. In addition, codon optimality is tightly correlated with both total and nuclear RNA levels, suggesting that codon usage broadly influences mRNA levels through transcription in a translation-independent manner. A large-scale RNA sequencing-based genetic screen in Neurospora identified 18 candidate factors that when deleted decreased the genome-wide correlation between codon usage and RNA levels and reduced the codon usage effect on gene expression. Most of these factors, such as the H3K36 methyltransferase, are chromatin regulators or transcription factors. Together, our results suggest that the transcriptional effect of codon usage is mediated by multiple transcriptional regulatory mechanisms.