Translation and mRNA Stability Control.

Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

mRNA stability and m6A are major determinants of subcellular mRNA localization in neurons.

For cells to perform their biological functions, they need to adopt specific shapes and form functionally distinct subcellular compartments. This is achieved in part via an asymmetric distribution of mRNAs within cells. Currently, the main model of mRNA localization involves specific sequences called "zipcodes" that direct mRNAs to their proper locations. However, while thousands of mRNAs localize within cells, only a few zipcodes have been identified, suggesting that additional mechanisms contribute to localization. Here, we assess the role of mRNA stability in localization by combining the isolation of the soma and neurites of mouse primary cortical and mESC-derived neurons, SLAM-seq, m6A-RIP-seq, the perturbation of mRNA destabilization mechanisms, and the analysis of multiple mRNA localization datasets. We show that depletion of mRNA destabilization elements, such as m6A, AU-rich elements, and suboptimal codons, functions as a mechanism that mediates the localization of mRNAs associated with housekeeping functions to neurites in several types of neurons.

Codon optimality-mediated mRNA degradation: Linking translational elongation to mRNA stability.

Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.

Translation elongation inhibitors stabilize select short-lived transcripts.

Translation elongation inhibitors are commonly used to study different cellular processes. Yet, their specific impact on transcription and mRNA decay has not been thoroughly assessed. Here we use TimeLapse sequencing to investigate how translational stress impacts mRNA dynamics in human cells. Our results reveal that a distinct group of transcripts is stabilized in response to the translation elongation inhibitor emetine. These stabilized mRNAs are short-lived at steady state and many of them encode C2H2 zinc finger proteins. The codon usage of these stabilized transcripts is suboptimal compared to other expressed transcripts, including other short-lived mRNAs that are not stabilized after emetine treatment. Finally, we show that stabilization of these transcripts is independent of ribosome quality control factors and signaling pathways activated by ribosome collisions. Our data describe a group of short-lived transcripts whose degradation is particularly sensitive to the inhibition of translation elongation.

Quantification of elongation stalls and impact on gene expression in yeast.

Ribosomal pauses are a critical part of cotranslational events including protein folding and localization. However, extended ribosome pauses can lead to ribosome collisions, resulting in the activation of ribosome rescue pathways and turnover of protein and mRNA. While this relationship has been known, there has been little exploration of how ribosomal stalls impact translation duration at a quantitative level. We have taken a method used to measure elongation time and adapted it for use in Saccharomyces cerevisiae to quantify the impact of elongation stalls. We find, in transcripts containing Arg CGA codon repeat-induced stalls, a Hel2-mediated dose-dependent decrease in protein expression and mRNA level and an elongation delay on the order of minutes. In transcripts that contain synonymous substitutions to nonoptimal Leu codons, there is a decrease in protein and mRNA levels, as well as similar elongation delay, but this occurs through a non-Hel2-mediated mechanism. Finally, we find that Dhh1 selectively increases protein expression, mRNA level, and elongation rate. This indicates that distinct poorly translated mRNAs will activate different rescue pathways despite similar elongation stall durations. Taken together, these results provide new quantitative mechanistic insight into the surveillance of translation and the roles of Hel2 and Dhh1 in mediating ribosome pausing events.

Dengue virus preferentially uses human and mosquito non-optimal codons.

Codon optimality refers to the effect that codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We demonstrated that codon optimality exists in mosquito cells and showed that DENV preferentially uses nonoptimal (destabilizing) codons and avoids codons that are defined as optimal (stabilizing) in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are upregulated during infection, and so is the tRNA decoding the nonoptimal and DENV preferentially used codon for arginine. We found that adaptation during single-host passaging in human or mosquito cells results in the selection of synonymous mutations towards DENV's preferred nonoptimal codons that increase virus fitness. Finally, our analyses revealed that hundreds of viruses preferentially use nonoptimal codons, with those infecting a single host displaying an even stronger bias, suggesting that host-pathogen interaction shapes virus-synonymous codon choice.

Cellular energy regulates mRNA degradation in a codon-specific manner.

Codon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. Here we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Mathematical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly alter the effect of codon usage. This new mode of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression.

The Fitness Effects of Codon Composition of the Horizontally Transferred Antibiotic Resistance Genes Intensify at Sub-lethal Antibiotic Levels.

The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5'-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5'-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes.

Natural Selection Shapes Codon Usage in the Human Genome.

Synonymous codon usage has been identified as a determinant of translational efficiency and mRNA stability in model organisms and human cell lines. However, whether natural selection shapes human codon content to optimize translation efficiency is unclear. Furthermore, aside from those that affect splicing, synonymous mutations are typically ignored as potential contributors to disease. Using genetic sequencing data from nearly 200,000 individuals, we uncover clear evidence that natural selection optimizes codon content in the human genome. In deriving intolerance metrics to quantify gene-level constraint on synonymous variation, we discover that dosage-sensitive genes, DNA-damage-response genes, and cell-cycle-regulated genes are particularly intolerant to synonymous variation. Notably, we illustrate that reductions in codon optimality in BRCA1 can attenuate its function. Our results reveal that synonymous mutations most likely play an underappreciated role in human variation.

FMRP links optimal codons to mRNA stability in neurons.

Fragile X syndrome (FXS) is caused by inactivation of the FMR1 gene and loss of encoded FMRP, an RNA binding protein that represses translation of some of its target transcripts. Here we use ribosome profiling and RNA sequencing to investigate the dysregulation of translation in the mouse brain cortex. We find that most changes in ribosome occupancy on hundreds of mRNAs are largely driven by dysregulation in transcript abundance. Many down-regulated mRNAs, which are mostly responsible for neuronal and synaptic functions, are highly enriched for FMRP binding targets. RNA metabolic labeling demonstrates that, in FMRP-deficient cortical neurons, mRNA down-regulation is caused by elevated degradation and is correlated with codon optimality. Moreover, FMRP preferentially binds mRNAs with optimal codons, suggesting that it stabilizes such transcripts through direct interactions via the translational machinery. Finally, we show that the paradigm of genetic rescue of FXS-like phenotypes in FMRP-deficient mice by deletion of the Cpeb1 gene is mediated by restoration of steady-state RNA levels and consequent rebalancing of translational homeostasis. Our data establish an essential role of FMRP in codon optimality-dependent mRNA stability as an important factor in FXS.

The effects of codon bias and optimality on mRNA and protein regulation.

The central dogma of molecular biology entails that genetic information is transferred from nucleic acid to proteins. Notwithstanding retro-transcribing genetic elements, DNA is transcribed to RNA which in turn is translated into proteins. Recent advancements have shown that each stage is regulated to control protein abundances for a variety of essential physiological processes. In this regard, mRNA regulation is essential in fine-tuning or calibrating protein abundances. In this review, we would like to discuss one of several mRNA-intrinsic features of mRNA regulation that has been gaining traction of recent-codon bias and optimality. Specifically, we address the effects of codon bias with regard to codon optimality in several biological processes centred on translation, such as mRNA stability and protein folding among others. Finally, we examine how different organisms or cell types, through this system, are able to coordinate physiological pathways to respond to a variety of stress or growth conditions.

Emerging Themes in Regulation of Global mRNA Turnover in cis.

mRNA is the molecule that conveys genetic information from DNA to the translation apparatus. mRNAs in all organisms display a wide range of stability, and mechanisms have evolved to selectively and differentially regulate individual mRNA stability in response to intracellular and extracellular cues. In recent years, three seemingly distinct aspects of RNA biology-mRNA N6-methyladenosine (m6A) modification, alternative 3' end processing and polyadenylation (APA), and mRNA codon usage-have been linked to mRNA turnover, and all three aspects function to regulate global mRNA stability in cis. Here, we discuss the discovery and molecular dissection of these mechanisms in relation to how they impact the intrinsic decay rate of mRNA in eukaryotes, leading to transcriptome reprogramming.

Codon bias confers stability to human mRNAs.

Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.

Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition.

Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.

RNA tales - how embryos read and discard messages from mom.

Following fertilization, embryos develop for a substantial amount of time with a transcriptionally silent genome. Thus, early development is maternally programmed, as it solely relies on RNAs and proteins that are provided by the female gamete. However, these maternal instructions are not sufficient to support later steps of embryogenesis and are therefore gradually replaced by novel products synthesized from the zygotic genome. This switch in the origin of molecular players that drive early development is known as the maternal-to-zygotic transition (MZT). MZT is a universal phenomenon among all metazoans and comprises two interconnected processes: maternal mRNA degradation and the transcriptional awakening of the zygotic genome. The recent adaptation of high-throughput methods for use in embryos has deepened our knowledge of the molecular principles underlying MZT. These mechanisms comprise conserved strategies for RNA regulation that operate in many well-studied cellular contexts but that have adapted differently to early development. In this Review, we will discuss advances in our understanding of post-transcriptional regulatory pathways that drive maternal mRNA clearance during MZT, with an emphasis on recent data in zebrafish embryos on codon-mediated mRNA decay, the contributions of microRNAs (miRNAs) and RNA-binding proteins to this process, and the roles of RNA modifications in the stability control of maternal mRNAs.

Translation elongation and mRNA stability are coupled through the ribosomal A-site.

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in eukaryotic cells. Previous work by us and others has shown that codon identity exerts a powerful influence on mRNA stability. In Saccharomyces cerevisiae, studies using a handful of reporter mRNAs show that optimal codons increase translation elongation rate, which in turn increases mRNA stability. However, a direct relationship between elongation rate and mRNA stability has not been established across the entire yeast transcriptome. In addition, there is evidence from work in higher eukaryotes that amino acid identity influences mRNA stability, raising the question as to whether the impact of translation elongation on mRNA decay is at the level of tRNA decoding, amino acid incorporation, or some combination of each. To address these questions, we performed ribosome profiling of wild-type yeast. In good agreement with other studies, our data showed faster codon-specific elongation over optimal codons and faster transcript-level elongation correlating with transcript optimality. At both the codon-level and transcript-level, faster elongation correlated with increased mRNA stability. These findings were reinforced by showing increased translation efficiency and kinetics for a panel of 11 HIS3 reporter mRNAs of increasing codon optimality. While we did observe that elongation measured by ribosome profiling is composed of both amino acid identity and synonymous codon effects, further analyses of these data establish that A-site tRNA decoding rather than other steps of translation elongation is driving mRNA decay in yeast.

A Universal Code for mRNA Stability?

Precise elimination of maternal mRNAs plays a critical role during the maternal-to-zygotic transition (MZT) to promote developmental processing. Two new studies demonstrate that, in eukaryotes, codon-mediated decay is a conserved mechanism to shape maternal mRNA stability by affecting deadenylation rate in a translation-dependent manner. These studies add to a growing body of literature suggesting that translational elongation rates are a major determinant of mRNA stability.

High-resolution profiling of NMD targets in yeast reveals translational fidelity as a basis for substrate selection.

Nonsense-mediated mRNA decay (NMD) plays an important role in eukaryotic gene expression, yet the scope and the defining features of NMD-targeted transcripts remain elusive. To address these issues, we reevaluated the genome-wide expression of annotated transcripts in yeast cells harboring deletions of the UPF1, UPF2, or UPF3 genes. Our new RNA-seq analyses confirm previous results of microarray studies, but also uncover hundreds of new NMD-regulated transcripts that had escaped previous detection, including many intron-containing pre-mRNAs and several noncoding RNAs. The vast majority of NMD-regulated transcripts are normal-looking protein-coding mRNAs. Our bioinformatics analyses reveal that this set of NMD-regulated transcripts generally have lower translational efficiency and higher ratios of out-of-frame translation. NMD-regulated transcripts also have lower average codon optimality scores and higher transition probability to nonoptimal codons. Collectively, our results generate a comprehensive catalog of yeast NMD substrates and yield new insights into the mechanisms by which these transcripts are targeted by NMD.

Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast.

The stability of mRNA is one of the major determinants of gene expression. Although a wealth of sequence elements regulating mRNA stability has been described, their quantitative contributions to half-life are unknown. Here, we built a quantitative model for Saccharomyces cerevisiae based on functional mRNA sequence features that explains 59% of the half-life variation between genes and predicts half-life at a median relative error of 30%. The model revealed a new destabilizing 3' UTR motif, ATATTC, which we functionally validated. Codon usage proves to be the major determinant of mRNA stability. Nonetheless, single-nucleotide variations have the largest effect when occurring on 3' UTR motifs or upstream AUGs. Analyzing mRNA half-life data of 34 knockout strains showed that the effect of codon usage not only requires functional decapping and deadenylation, but also the 5'-to-3' exonuclease Xrn1, the nonsense-mediated decay genes, but not no-go decay. Altogether, this study quantitatively delineates the contributions of mRNA sequence features on stability in yeast, reveals their functional dependencies on degradation pathways, and allows accurate prediction of half-life from mRNA sequence.