Advances in methods for tRNA sequencing and quantification.

In the past decade tRNA sequencing (tRNA-seq) has attracted considerable attention as an important tool for the development of novel approaches to quantify highly modified tRNA species and to propel tRNA research aimed at understanding the cellular physiology and disease and development of tRNA-based therapeutics. Many methods are available to quantify tRNA abundance while accounting for modifications and tRNA charging/acylation. Advances in both library preparation methods and bioinformatic workflows have enabled developments in next-generation sequencing (NGS) workflows. Other approaches forgo NGS applications in favor of hybridization-based approaches. In this review we provide a brief comparative overview of various tRNA quantification approaches, focusing on the advantages and disadvantages of these methods, which together facilitate reliable tRNA quantification.

Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2.

GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNAPhe, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.

A Unique ISR Program Determines Cellular Responses to Chronic Stress.

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

Synonymous Codons Direct Cotranslational Folding toward Different Protein Conformations.

In all genomes, most amino acids are encoded by more than one codon. Synonymous codons can modulate protein production and folding, but the mechanism connecting codon usage to protein homeostasis is not known. Here we show that synonymous codon variants in the gene encoding gamma-B crystallin, a mammalian eye-lens protein, modulate the rates of translation and cotranslational folding of protein domains monitored in real time by Förster resonance energy transfer and fluorescence-intensity changes. Gamma-B crystallins produced from mRNAs with changed codon bias have the same amino acid sequence but attain different conformations, as indicated by altered in vivo stability and in vitro protease resistance. 2D NMR spectroscopic data suggest that structural differences are associated with different cysteine oxidation states of the purified proteins, providing a link between translation, folding, and the structures of isolated proteins. Thus, synonymous codons provide a secondary code for protein folding in the cell.

A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.

Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

Translation initiation on mammalian mRNAs with structured 5'UTRs requires DExH-box protein DHX29.

Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.

Molecular Peptide Grafting as a Tool to Create Novel Protein Therapeutics.

The study of peptides (synthetic or corresponding to discrete regions of proteins) has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, the functional activity of many short peptides is usually substantially lower than that of their parental proteins. This is (as a rule) due to their diminished structural organization, stability, and solubility often leading to an enhanced propensity for aggregation. Several approaches have emerged to overcome these limitations, which are aimed at imposing structural constraints into the backbone and/or sidechains of the therapeutic peptides (such as molecular stapling, peptide backbone circularization and molecular grafting), therefore enforcing their biologically active conformation and thus improving their solubility, stability, and functional activity. This review provides a short summary of approaches aimed at enhancing the biological activity of short functional peptides with a particular focus on the peptide grafting approach, whereby a functional peptide is inserted into a scaffold molecule. Intra-backbone insertions of short therapeutic peptides into scaffold proteins have been shown to enhance their activity and render them a more stable and biologically active conformation.

eIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

Mutually exclusive amino acid residues of L13a are responsible for its ribosomal incorporation and translational silencing leading to resolution of inflammation.

Eukaryotic ribosomal protein L13a is a member of the conserved universal ribosomal uL13 protein family. Structurally, L13a is distinguished from its prokaryotic counterparts by the presence of an ∼55 amino acid-long carboxy-terminal α-helical extension. The importance of these evolved residues in the carboxy-terminal extension for mammalian ribosome biogenesis as well as L13a's extraribosomal function in GAIT (γ interferon-activated inhibitor of translation) complex-mediated translation silencing during inflammation is not understood. Here, we present biochemical analyses of L13a mutant variants identifying several mutually exclusive amino acid residues in the eukaryote-specific carboxy-terminal extension of human L13a (Tyr149-Val203) important for ribosomal incorporation and translational silencing. Specifically, we show that mutation of Arg169, Lys170, and Lys171 to Ala abrogate GAIT-mediated translational silencing, but not L13a incorporation into ribosomes. Moreover, we show that the carboxy-terminal helix alone can silence translation of GAIT element-containing mRNAs in vitro. We also show through cellular immunofluorescence experiments that nuclear but not nucleolar localization of L13a is resistant to extensive amino acid alterations, suggesting that multiple complex nuclear import signals are present within this protein. These studies provide new insights into L13a structure and its ribosomal and extraribosomal functions in model human cells.

Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.

Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2α; P-EIF2α), active DNA damage checkpoints, and cell-cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a 'stress-dependent' translation profile. This includes further elevated P-eIF2α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

A Code Within a Code: How Codons Fine-Tune Protein Folding in the Cell.

The genetic code sets the correspondence between the sequence of a given nucleotide triplet in an mRNA molecule, called a codon, and the amino acid that is added to the growing polypeptide chain during protein synthesis. With four bases (A, G, U, and C), there are 64 possible triplet codons: 61 sense codons (encoding amino acids) and 3 nonsense codons (so-called, stop codons that define termination of translation). In most organisms, there are 20 common/standard amino acids used in protein synthesis; thus, the genetic code is redundant with most amino acids (with the exception of Met and Trp) are being encoded by more than one (synonymous) codon. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in mRNA suggested that the specific codon choice might have functional implications beyond coding for amino acid. Observation of nonequivalent use of codons in mRNAs implied a possibility of the existence of auxiliary information in the genetic code. Indeed, it has been found that genetic code contains several layers of such additional information and that synonymous codons are strategically placed within mRNAs to ensure a particular translation kinetics facilitating and fine-tuning co-translational protein folding in the cell via step-wise/sequential structuring of distinct regions of the polypeptide chain emerging from the ribosome at different points in time. This review summarizes key findings in the field that have identified the role of synonymous codons and their usage in protein folding in the cell.

Role of the uS9/yS16 C-terminal tail in translation initiation and elongation in Saccharomyces cerevisiae.

The small ribosomal subunit protein uS9 (formerly called rpS16 in Saccharomyces cerevisiae), has a long protruding C-terminal tail (CTT) that extends towards the mRNA cleft of the ribosome. The last C-terminal residue of uS9 is an invariably conserved, positively charged Arg that is believed to enhance interaction of the negatively charged initiator tRNA with the ribosome when the tRNA is base-paired to the AUG codon in the P-site. In order to more fully characterize the role of the uS9 CTT in eukaryotic translation, we tested how truncations, extensions and substitutions within the CTT affect initiation and elongation processes in Saccharomyces cerevisiae. We found that uS9 C-terminal residues are critical for efficient recruitment of the eIF2•GTP•Met-tRNAiMet ternary complex to the ribosome and for its proper response to the presence of an AUG codon in the P-site during the scanning phase of initiation. These residues also regulate hydrolysis of the GTP in the eIF2•GTP•Met-tRNAiMet complex to GDP and Pi. In addition, our data show that uS9 CTT modulates elongation fidelity. Therefore, we propose that uS9 CTT is critical for proper control of the complex interplay of events surrounding accommodation of initiator and elongator tRNAs in the P- and A-sites of the ribosome.

Non-canonical role of cancer-associated mutant SEC23B in the ribosome biogenesis pathway.

SEC23B is a component of coat protein complex II (COPII) vesicles that transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Loss-of-function SEC23B mutations cause a rare form of anemia, resulting from decreased SEC23B levels. We recently identified germline heterozygous SEC23B variants as potentially cancer-predisposing. Mutant SEC23B associated with ER stress-mediated tumorigenesis, without decreased SEC23B expression. However, our understanding of the processes behind these observations remain limited. Here, we show mutant SEC23B exists within nucleoli, in addition to classical distribution at the ER/Golgi. This occurs independent of other COPII proteins and does not compromise secretory function. Mutant cells have increased ribosomal protein and translation-related gene expression, and enhanced translational capacity, in the presence of ER stress. We show that mutant SEC23B binds to UBF transcription factor, with increased UBF transcription factor binding at the ribosomal DNA promoter. Our data indicate SEC23B has potential non-canonical COPII-independent function, particularly within the ribosome biogenesis pathway, and that may contribute to the pathogenesis of cancer-predisposition.

A Retrospective on eIF2A-and Not the Alpha Subunit of eIF2.

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,ß,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

Unraveling co-translational protein folding: Concepts and methods.

Advances in techniques such as nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and single-molecule and time-resolved fluorescent approaches are transforming our ability to study co-translational protein folding both in vivo in living cells and in vitro in reconstituted cell-free translation systems. These approaches provide comprehensive information on the spatial organization and dynamics of nascent polypeptide chains and the kinetics of co-translational protein folding. This information has led to an improved understanding of the process of protein folding in living cells and should allow remaining key questions in the field, such as what structures are formed within nascent chains during protein synthesis and when, to be answered. Ultimately, studies using these techniques will facilitate development of a unified concept of protein folding, a process that is essential for proper cell function and organism viability. This review describes current methods for analysis of co-translational protein folding with an emphasis on some of the recently developed techniques that allow monitoring of co-translational protein folding in real-time.

Conserved structures formed by heterogeneous RNA sequences drive silencing of an inflammation responsive post-transcriptional operon.

RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation.

A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity.

Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure.

The Yin and Yang of codon usage.

The genetic code is degenerate. With the exception of two amino acids (Met and Trp), all other amino acid residues are each encoded by multiple, so-called synonymous codons. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in genes/genomes suggested that codon choice might have functional implications beyond amino acid coding. The pattern of non-uniform codon use (known as codon usage bias) varies between organisms and represents a unique feature of an organism. Organism-specific codon choice is related to organism-specific differences in populations of cognate tRNAs. This implies that, in a given organism, frequently used codons will be translated more rapidly than infrequently used ones and vice versa A theory of codon-tRNA co-evolution (necessary to balance accurate and efficient protein production) was put forward to explain the existence of codon usage bias. This model suggests that selection favours preferred (frequent) over un-preferred (rare) codons in order to sustain efficient protein production in cells and that a given un-preferred codon will have the same effect on an organism's fitness regardless of its position within an mRNA's open reading frame. However, many recent studies refute this prediction. Un-preferred codons have been found to have important functional roles and their effects appeared to be position-dependent. Synonymous codon usage affects the efficiency/stringency of mRNA decoding, mRNA biogenesis/stability, and protein secretion and folding. This review summarizes recent developments in the field that have identified novel functions of synonymous codons and their usage.