Repertoires of tRNAs: The Couplers of Genomics and Proteomics.

The pool of transfer RNA (tRNA) molecules in cells allows the ribosome to decode genetic information. This repertoire of molecular decoders is positioned in the crossroad of the genome, the transcriptome, and the proteome. Omics and systems biology now allow scientists to explore the entire repertoire of tRNAs of many organisms, revealing basic exciting biology. The tRNA gene set of hundreds of species is now characterized, in addition to the tRNA genes of organelles and viruses. Genes encoding tRNAs for certain anticodon types appear in dozens of copies in a genome, while others are universally absent from any genome. Transcriptome measurement of tRNAs is challenging, but in recent years new technologies have allowed researchers to determine the dynamic expression patterns of tRNAs. These advances reveal that availability of ready-to-translate tRNA molecules is highly controlled by several transcriptional and posttranscriptional regulatory processes. This regulation shapes the proteome according to the cellular state. The tRNA pool profoundly impacts many aspects of cellular and organismal life, including protein expression level, translation accuracy, adequacy of folding, and even mRNA stability. As a result, the shape of the tRNA pool affects organismal health and may participate in causing conditions such as cancer and neurological conditions.

Structural mechanism of angiogenin activation by the ribosome.

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

mRNA reading frame maintenance during eukaryotic ribosome translocation.

One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.

Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

Accuracy mechanism of eukaryotic ribosome translocation.

Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon-anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon-anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a 'pawl' defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation.

Systematic Detection of Amino Acid Substitutions in Proteomes Reveals Mechanistic Basis of Ribosome Errors and Selection for Translation Fidelity.

The translation machinery and the genes it decodes co-evolved to achieve production throughput and accuracy. Nonetheless, translation errors are frequent, and they affect physiology and protein evolution. Mapping translation errors in proteomes and understanding their causes is hindered by lack of a proteome-wide experimental methodology. We present the first methodology for systematic detection and quantification of errors in entire proteomes. Following proteome mass spectrometry, we identify, in E. coli and yeast, peptides whose mass indicates specific amino acid substitutions. Most substitutions result from codon-anticodon mispairing. Errors occur at sites that evolve rapidly and that minimally affect energetic stability, indicating selection for high translation fidelity. Ribosome density data show that errors occur at sites where ribosome velocity is higher, demonstrating a trade-off between speed and accuracy. Treating bacteria with an aminoglycoside antibiotic or deprivation of specific amino acids resulted in particular patterns of errors. These results reveal a mechanistic and evolutionary basis for translation fidelity.

Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

Contribution of tRNA sequence and modifications to the decoding preferences of E. coli and M. mycoides tRNAGlyUCC for synonymous glycine codons.

tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding.

An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

Fine-tuning the tRNA anticodon arm for multiple/consecutive incorporations of ß-amino acids and analogs.

Ribosomal incorporation of ß-amino acids into nascent peptides is much less efficient than that of the canonical α-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved ß-amino acid incorporation. However, multiple/consecutive incorporations of ß-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of ß-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of ß-homophenylglycine (ßPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive ßPhg's. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for ß-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of ß-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple ß-amino acids.

Cell type-specific analysis by single-cell profiling identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons.

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using available mouse and human single-cell RNA-sequencing atlases. After showing the robustness of ATAC-sequencing measurements for the analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in both mouse and human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons, which clustered separately from other cell types. Integration of these data sets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell type-specific ribosome profiling, suggesting that the reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency resolved at the cell-type level with single-cell information, identifies a conserved landscape of translation elongation across mammalian cellular diversity and evolution.

Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon.

Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium Salmonella enterica serovar Typhimurium, 5' tRNA halves ending in 2',3' cyclic phosphate are proposed to bind the RtcR transcriptional activator, resulting in transcription of an RNA repair operon. However, since 5' and 3' tRNA halves can remain base paired after cleavage, the 5' tRNA halves could potentially bind RtcR as nicked tRNAs. Here we report that nicked tRNAs are ligands for RtcR. By isolating RNA from bacteria under conditions that preserve base pairing, we show that many tRNA halves are in the form of nicked tRNAs. Using a circularly permuted tRNA that mimics a nicked tRNA, we show that nicked tRNA ending in 2',3' cyclic phosphate is a better ligand for RtcR than the corresponding 5' tRNA half. In human cells, we show that some tRNA halves similarly remain base paired as nicked tRNAs following cleavage by anticodon nucleases. Our work supports a role for the RNA repair operon in repairing nicked tRNAs and has implications for the functions proposed for tRNA fragments in eukaryotes.

Drop-off-reinitiation at the amino termini of nascent peptides and its regulation by IF3, EF-G, and RRF.

In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the anticodon of fMet-tRNAini and then relocates the fMet-tRNAini to an active position. Here, we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of the codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson-Crick, are more preferred, but other types of base pairs, for example, G/U wobble, are also tolerated; weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNAini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the amino-terminal fMet. We refer to this event as the amino-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNAini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the amino-terminal drop-off-reinitiation.

Posttranscriptional modification to the core of tRNAs modulates translational misreading errors.

Protein synthesis on the ribosome involves successive rapid recruitment of cognate aminoacyl-tRNAs and rejection of the much more numerous incorrect near- or non-cognates. The principal feature of translation elongation is that at every step, many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translational accuracy require that cognate tRNAs have relatively similar acceptance rates by the ribosome. To achieve that, tRNAs evolved to compensate for differences in amino acid properties and codon-anticodon strength that affect acceptance. Part of that response involved tRNA posttranscriptional modifications, which can affect tRNA decoding efficiency, accuracy, and structural stability. The most intensively modified regions of the tRNA are the anticodon loop and structural core of the tRNA. Anticodon loop modifications directly affect codon-anticodon pairing and therefore modulate accuracy. Core modifications have been thought to ensure consistent decoding rates principally by stabilizing tRNA structure to avoid degradation; however, degradation due to instability appears to only be a significant issue above normal growth temperatures. We suspected that the greater role of modification at normal temperatures might be to tune tRNAs to maintain consistent intrinsic rates of acceptance and peptide transfer and that hypomodification by altering these rates might degrade the process of discrimination, leading to increased translational errors. Here, we present evidence that most tRNA core modifications do modulate the frequency of misreading errors, suggesting that the need to maintain accuracy explains their deep evolutionary conservation.

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing.

Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.

Structural basis for reduced ribosomal A-site fidelity in response to P-site codon-anticodon mismatches.

Rapid and accurate translation is essential in all organisms to produce properly folded and functional proteins. mRNA codons that define the protein-coding sequences are decoded by tRNAs on the ribosome in the aminoacyl (A) binding site. The mRNA codon and the tRNA anticodon interaction is extensively monitored by the ribosome to ensure accuracy in tRNA selection. While other polymerases that synthesize DNA and RNA can correct for misincorporations, the ribosome is unable to correct mistakes. Instead, when a misincorporation occurs, the mismatched tRNA-mRNA pair moves to the peptidyl (P) site and, from this location, causes a reduction in the fidelity at the A site, triggering post-peptidyl transfer quality control. This reduced fidelity allows for additional incorrect tRNAs to be accepted and for release factor 2 (RF2) to recognize sense codons, leading to hydrolysis of the aberrant peptide. Here, we present crystal structures of the ribosome containing a tRNALys in the P site with a U•U mismatch with the mRNA codon. We find that when the mismatch occurs in the second position of the P-site codon-anticodon interaction, the first nucleotide of the A-site codon flips from the mRNA path to engage highly conserved 16S rRNA nucleotide A1493 in the decoding center. We propose that this mRNA nucleotide mispositioning leads to reduced fidelity at the A site. Further, this state may provide an opportunity for RF2 to initiate premature termination before erroneous nascent chains disrupt the cellular proteome.

Initial Amino Acid:Codon Assignments and Strength of Codon:Anticodon Binding.

The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.

Molecular Coping Mechanisms: Reprogramming tRNAs To Regulate Codon-Biased Translation of Stress Response Proteins.

As part of the classic central dogma of molecular biology, transfer RNAs (tRNAs) are integral to protein translation as the adaptor molecules that link the genetic code in messenger RNA (mRNA) to the amino acids in the growing peptide chain. tRNA function is complicated by the existence of 61 codons to specify 20 amino acids, with most amino acids coded by two or more synonymous codons. Further, there are often fewer tRNAs with unique anticodons than there are synonymous codons for an amino acid, with a single anticodon able to decode several codons by "wobbling" of the base pairs arising between the third base of the codon and the first position on the anticodon. The complications introduced by synonymous codons and wobble base pairing began to resolve in the 1960s with the discovery of dozens of chemical modifications of the ribonucleotides in tRNA, which, by analogy to the epigenome, are now collectively referred to as the epitranscriptome for not changing the genetic code inherent to all RNA sequences. tRNA modifications were found to stabilize codon-anticodon interactions, prevent misinitiation of translation, and promote translational fidelity, among other functions, with modification deficiencies causing pathological phenotypes. This led to hypotheses that modification-dependent tRNA decoding efficiencies might play regulatory roles in cells. However, it was only with the advent of systems biology and convergent "omic" technologies that the higher level function of synonymous codons and tRNA modifications began to emerge.Here, we describe our laboratories' discovery of tRNA reprogramming and codon-biased translation as a mechanism linking tRNA modifications and synonymous codon usage to regulation of gene expression at the level of translation. Taking a historical approach, we recount how we discovered that the 8-10 modifications in each tRNA molecule undergo unique reprogramming in response to cellular stresses to promote translation of mRNA transcripts with unique codon usage patterns. These modification tunable transcripts (MoTTs) are enriched with specific codons that are differentially decoded by modified tRNAs and that fall into functional families of genes encoding proteins necessary to survive the specific stress. By developing and applying systems-level technologies, we showed that cells lacking specific tRNA modifications are sensitized to certain cellular stresses by mistranslation of proteins, disruption of mitochondrial function, and failure to translate critical stress response proteins. In essence, tRNA reprogramming serves as a cellular coping strategy, enabling rapid translation of proteins required for stress-specific cell response programs. Notably, this phenomenon has now been characterized in all organisms from viruses to humans and in response to all types of environmental changes. We also elaborate on recent findings that cancer cells hijack this mechanism to promote their own growth, metastasis, and chemotherapeutic resistance. We close by discussing how understanding of codon-biased translation in various systems can be exploited to develop new therapeutics and biomanufacturing processes.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.