Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Ribosome slowdown triggers codon-mediated mRNA decay independently of ribosome quality control.

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role for the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analyses are required to clarify the codon effects on mRNA stability and the related cotranslational mRNA decay pathways, for example, those triggered by aberrant ribosome stalling. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of the ribosome quality control factor Znf598 and stalling-dependent mRNA decay. We propose that codon-mediated mRNA decay is rather triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

Origin and Evolution of the Universal Genetic Code.

The standard genetic code (SGC) is virtually universal among extant life forms. Although many deviations from the universal code exist, particularly in organelles and prokaryotes with small genomes, they are limited in scope and obviously secondary. The universality of the code likely results from the combination of a frozen accident, i.e., the deleterious effect of codon reassignment in the SGC, and the inhibitory effect of changes in the code on horizontal gene transfer. The structure of the SGC is nonrandom and ensures high robustness of the code to mutational and translational errors. However, this error minimization is most likely a by-product of the primordial code expansion driven by the diversification of the repertoire of protein amino acids, rather than a direct result of selection. Phylogenetic analysis of translation system components, in particular aminoacyl-tRNA synthetases, shows that, at a stage of evolution when the translation system had already attained high fidelity, the correspondence between amino acids and cognate codons was determined by recognition of amino acids by RNA molecules, i.e., proto-tRNAs. We propose an experimentally testable scenario for the evolution of the code that combines recognition of amino acids by unique sites on proto-tRNAs (distinct from the anticodons), expansion of the code via proto-tRNA duplication, and frozen accident.

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Stop or Not: Genome-Wide Profiling of Reassigned Stop Codons in Ciliates.

Bifunctional stop codons that have both translation and termination functions in the same species are important for understanding the evolution and function of genetic codes in living organisms. Considering the high frequency of bifunctional codons but limited number of available genomes in ciliates, we de novo sequenced seven representative ciliate genomes to explore the evolutionary history of stop codons. We further propose a stop codon reassignment quantification method (stopCR) that can identify bifunctional codons and measure their frequencies in various eukaryotic organisms. Using our newly developed method, we found two previously undescribed genetic codes, illustrating the prevalence of bifunctional stop codons in ciliates. Overall, evolutionary genomic analyses suggest that gain or loss of reassigned stop codons in ciliates is shaped by their living environment, the eukaryotic release factor 1, and suppressor tRNAs. This study provides novel clues about the functional diversity and evolutionary history of stop codons in eukaryotic organisms.

The Branched Nature of the Nonsense-Mediated mRNA Decay Pathway.

Nonsense-mediated mRNA decay (NMD) is a conserved translation-coupled quality control mechanism in all eukaryotes that regulates the expression of a significant fraction of both the aberrant and normal transcriptomes. In vertebrates, NMD has become an essential process owing to expansion of the diversity of NMD-regulated transcripts, particularly during various developmental processes. Surprisingly, however, some core NMD factors that are essential for NMD in simpler organisms appear to be dispensable for vertebrate NMD. At the same time, numerous NMD enhancers and suppressors have been identified in multicellular organisms including vertebrates. Collectively, the available data suggest that vertebrate NMD is a complex, branched pathway wherein individual branches regulate specific mRNA subsets to fulfill distinct physiological functions.

Codon usage bias and phylogenetic analysis of chloroplast genome in 36 gracilariaceae species.

Gracilariaceae is a group of marine large red algae and main source of agar with important economic and ecological value. The codon usage patterns of chloroplast genomes in 36 species from Graciliaceae show that GC range from 0.284 to 0.335, the average GC3 range from 0.135 to 0.243 and the value of ENC range from 35.098 to 42.327, which indicates these genomes are rich in AT and prefer to use codons ending with AT in these species. Nc plot, PR2 plot, neutrality plot analyses and correlation analysis indicate that these biases may be caused by multiple factors, such as natural selection and mutation pressure, but prolonged natural selection is the main driving force influencing codon usage preference. The cluster analysis and phylogenetic analysis show that the differentiation relationship of them is different and indicate that codons with weak or unbiased preferences may also play an irreplaceable role in these species' evolution. In addition, we identified 26 common high-frequency codons and 8-18 optimal codons all ending in A/U in these 36 species. Our results will not only contribute to carrying out transgenic work in Gracilariaceae species to maximize the protein yield in the future, but also lay a theoretical foundation for further exploring systematic classification of them.

Modifications of the 5' region of the CASPONTM tag's mRNA further enhance soluble recombinant protein production in Escherichia coli.

BACKGROUND: Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. RESULTS: In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6 kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. CONCLUSIONS: This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPONTM tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Combinations of slow-translating codon clusters can increase mRNA half-life in Saccharomyces cerevisiae.

The presence of a single cluster of nonoptimal codons was found to decrease a transcript's half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae The impact of multiple nonoptimal codon clusters on a transcript's half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5' end can lead to synergistic effects that increase a messenger RNA's (mRNA's) half-life in S. cerevisiae Specifically, the 5' end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5' end is the primary determinant of mRNA half-life. These results suggest the "translational ramp," in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes.

Selection of the translation initiation site (TIS) is a crucial step during translation. In the 1980s Marylin Kozak performed key studies on vertebrate mRNAs to characterize the optimal TIS consensus sequence, the Kozak motif. Within this motif, conservation of nucleotides in crucial positions, namely a purine at -3 and a G at +4 (where the A of the AUG is numbered +1), is essential for TIS recognition. Ever since its characterization the Kozak motif has been regarded as the optimal sequence to initiate translation in all eukaryotes. We revisit here published in silico data on TIS consensus sequences, as well as experimental studies from diverse eukaryotic lineages, and propose that, while the -3A/G position is universally conserved, the remaining variability of the consensus sequences enables their classification as optimal, strong, and moderate TIS sequences.

Mechanisms governing codon usage bias and the implications for protein expression in the chloroplast of Chlamydomonas reinhardtii.

Chloroplasts possess a considerably reduced genome that is decoded via an almost minimal set of tRNAs. These features make an excellent platform for gaining insights into fundamental mechanisms that govern protein expression. Here, we present a comprehensive and revised perspective of the mechanisms that drive codon selection in the chloroplast of Chlamydomonas reinhardtii and the functional consequences for protein expression. In order to extract this information, we applied several codon usage descriptors to genes with different expression levels. We show that highly expressed genes strongly favor translationally optimal codons, while genes with lower functional importance are rather affected by directional mutational bias. We demonstrate that codon optimality can be deduced from codon-anticodon pairing affinity and, for a small number of amino acids (leucine, arginine, serine, and isoleucine), tRNA concentrations. Finally, we review, analyze, and expand on the impact of codon usage on protein yield, secondary structures of mRNA, translation initiation and termination, and amino acid composition of proteins, as well as cotranslational protein folding. The comprehensive analysis of codon choice provides crucial insights into heterologous gene expression in the chloroplast of C. reinhardtii, which may also be applicable to other chloroplast-containing organisms and bacteria.

Codon usage pattern of the ancestor of green plants revealed through Rhodophyta.

Rhodophyta are among the closest known relatives of green plants. Studying the codons of their genomes can help us understand the codon usage pattern and characteristics of the ancestor of green plants. By studying the codon usage pattern of all available red algae, it was found that although there are some differences among species, high-bias genes in most red algae prefer codons ending with GC. Correlation analysis, Nc-GC3s plots, parity rule 2 plots, neutrality plot analysis, differential protein region analysis and comparison of the nucleotide content of introns and flanking sequences showed that the bias phenomenon is likely to be influenced by local mutation pressure and natural selection, the latter of which is the dominant factor in terms of translation accuracy and efficiency. It is worth noting that selection on translation accuracy could even be detected in the low-bias genes of individual species. In addition, we identified 15 common optimal codons in seven red algae except for G. sulphuraria for the first time, most of which were found to be complementary and bound to the tRNA genes with the highest copy number. Interestingly, tRNA modification was found for the highly degenerate amino acids of all multicellular red algae and individual unicellular red algae, which indicates that highly biased genes tend to use modified tRNA in translation. Our research not only lays a foundation for exploring the characteristics of codon usage of the red algae as green plant ancestors, but will also facilitate the design and performance of transgenic work in some economic red algae in the future.

The synthetic aminoglycoside ELX-02 induces readthrough of G550X-CFTR producing superfunctional protein that can be further enhanced by CFTR modulators.

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

The Codon Statistics Database: A Database of Codon Usage Bias.

We present the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq (over 15,000). The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage, and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons, Codon Adaptation Index, and frequency of optimal codons. Equivalent tables can be accessed for (1) all nuclear genes, (2) nuclear genes encoding ribosomal proteins, (3) mitochondrial genes, and (4) chloroplast genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., "primates") and obtain a table comparing all the species in the group. The database is free to access without registration at http://codonstatsdb.unr.edu.

Variation in Release Factor Abundance Is Not Needed to Explain Trends in Bacterial Stop Codon Usage.

In bacteria stop codons are recognized by one of two class I release factors (RF1) recognizing TAG, RF2 recognizing TGA, and TAA being recognized by both. Variation across bacteria in the relative abundance of RF1 and RF2 is thus hypothesized to select for different TGA/TAG usage. This has been supported by correlations between TAG:TGA ratios and RF1:RF2 ratios across multiple bacterial species, potentially also explaining why TAG usage is approximately constant despite extensive variation in GC content. It is, however, possible that stop codon trends are determined by other forces and that RF ratios adapt to stop codon usage, rather than vice versa. Here, we determine which direction of the causal arrow is the more parsimonious. Our results support the notion that RF1/RF2 ratios become adapted to stop codon usage as the same trends, notably the anomalous TAG behavior, are seen in contexts where RF1:RF2 ratios cannot be, or are unlikely to be, causative, that is, at 3'untranslated sites never used for translation termination, in intragenomic analyses, and across archaeal species (that possess only one RF1). We conclude that specifics of RF biology are unlikely to fully explain TGA/TAG relative usage. We discuss why the causal relationships for the evolution of synonymous stop codon usage might be different from those affecting synonymous sense codon usage, noting that transitions between TGA and TAG require two-point mutations one of which is likely to be deleterious.

Non-random Codon Usage of Synonymous and Non-synonymous Mutations in the Human HLA-A Gene.

The structure and function of human leucocyte antigen (HLA-A) is well known and is an extremely variable protein. From the public HLA-A database, we chose 26 high frequency HLA-A alleles (45% of sequenced alleles). Using five arbitrary references from these alleles, we analyzed synonymous mutations at the third codon position (sSNP3) and non-synonymous mutations (NSM). Both mutation types showed non-random locations of 29 sSNP3 codons and 71 NSM codons in the five reference lists. Most sSNP3 codons show identical mutation types with many mutations resulting from cytosine deamination. We proposed 23 ancestral parents of sSNP3 in five reference sequences using conserved parents in five unidirectional codons and 18 majority parents in reciprocal codons. These 23 proposed ancestral parents show exclusive codon usage of G3 or C3 parents located on both DNA strands that mutate to A3 or T3 variants mostly (76%) by cytosine deamination The sSNP3 and NSM show clear separation of the two variant types with most sSNP3 located in conserved areas in exons 2, 3 and 4, compared to most NSM appearing in two Variable Areas with no sSNP3 in the latter parts of exons 2 (α1) and 3 (α2). The Variable Areas contain NSM (polymorphic) residues at the center of the groove that bind the foreign peptide. We find distinctly different mutation patterns in NSM codons from those of sSNP3. Namely, G-C to A-T mutation frequency was much smaller, suggesting that evolutional pressures of deamination and other mechanisms applied to the two areas are significantly different.

Translation termination in human mitochondria - substrate specificity of mitochondrial release factors.

Mitochondria are the essential players in eukaryotic ATP production by oxidative phosphorylation, which relies on the maintenance and accurate expression of the mitochondrial genome. Even though the basic principles of translation are conserved due to the descendance from a bacterial ancestor, some deviations regarding translation factors as well as mRNA characteristics and the applied genetic code are present in human mitochondria. Together, these features are certain challenges during translation the mitochondrion has to handle. Here, we discuss the current knowledge regarding mitochondrial translation focusing on the termination process and the associated quality control mechanisms. We describe how mtRF1a resembles bacterial RF1 mechanistically and summarize in vitro and recent in vivo data leading to the conclusion of mtRF1a being the major mitochondrial release factor. On the other hand, we discuss the ongoing debate about the function of the second codon-dependent mitochondrial release factor mtRF1 regarding its role as a specialized termination factor. Finally, we link defects in mitochondrial translation termination to the activation of mitochondrial rescue mechanisms highlighting the importance of ribosome-associated quality control for sufficient respiratory function and therefore for human health.

Factors Affecting Readthrough of Natural Versus Premature Termination Codons.

Nonsense mutations occur within the open-reading frame of a gene resulting in a premature termination codon (PTC). PTC-containing mRNAs can either be degeraded or cause premature translation termination producing a truncated protein that can be either nonfunctional or toxic. Translational readthrough inducing drugs (TRIDs) are small molecules that are able to induce readthrough, resulting in the restoration of full-length protein expression. The re-expressed proteins usually harbor a missense change. The effciency of individual TRIDs is variable and varies between different genes and even different nonsense mutations in the same gene. This review summarizes factors, including the sequences located upstream and downstream the disease-causing mutation and the type of PTC, affecting the translational readthrough process by modulating the type of amino acid insertion and the efficiency of the process during readthrough following TRIDs treatments.

Evolution of the genetic code; Evidence from serine codon use disparity in Escherichia coli.

Among the 20 amino acids, three of them-leucine (Leu), arginine (Arg), and serine (Ser)-are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of the Escherichia coli genome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.