The epitranscriptome includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m3C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/2B-, METTL6-, and METTL2A/2B/6-dependent m3C profiles in human cells. METTL2A/2B modifies tRNA-arginine and tRNA-threonine members, whereas METTL6 modifies the tRNA-serine family. However, decreased m3C32 on tRNA-Ser-GCT isodecoders is only observed with combined METTL2A/2B/6 deletion. Ribo-Seq reveals altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed altered cell cycle, slowed proliferation, and increased cisplatin sensitivity phenotypes of METTL2A/2B/6-deficient cells. Thus, we define METTL2A/2B/6-dependent methylomes and uncover a particular requirement of m3C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes.

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons, which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3\') by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosome\'s A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5\'-NNN GNN codon pairs is likely mediated, in part, by the ribosome\'s CAR surface, which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5\'-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences.

The genetic code consists of 61 codons coding for 20 amino acids. These codons are recognized by transfer RNAs (tRNAs) that bind to specific codons during protein synthesis. All organisms utilize less than all 61 possible anticodons due to base pair wobble: the ability to have a mismatch with a codon at its third nucleotide. Previous studies observed a correlation between the tRNA pool of bacteria and the temperature of their respective environments. However, it is unclear if these patterns represent biological adaptations to maintain the efficiency and accuracy of protein synthesis in different environments. A mechanistic mathematical model of mRNA translation is used to quantify the expected elongation rates and error rate for each codon based on an organism\'s tRNA pool. A comparative analysis across a range of bacteria that accounts for covariance due to shared ancestry is performed to quantify the impact of environmental temperature on the evolution of the tRNA pool. We find that thermophiles generally have more anticodons represented in their tRNA pool than mesophiles or psychrophiles. Based on our model, this increased diversity is expected to lead to increased missense errors. The implications of this for protein evolution in thermophiles are discussed.

The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.

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.

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.

I analyzed all the theories and models of the origin of the genetic code, and over the years, I have considered the main suggestions that could explain this origin. The conclusion of this analysis is that the coevolution theory of the origin of the genetic code is the theory that best captures the majority of observations concerning the organization of the genetic code. In other words, the biosynthetic relationships between amino acids would have heavily influenced the origin of the organization of the genetic code, as supported by the coevolution theory. Instead, the presence in the genetic code of physicochemical properties of amino acids, which have also been linked to the physicochemical properties of anticodons or codons or bases by stereochemical and physicochemical theories, would simply be the result of natural selection. More explicitly, I maintain that these correlations between codons, anticodons or bases and amino acids are in fact the result not of a real correlation between amino acids and codons, for example, but are only the effect of the intervention of natural selection. Specifically, in the genetic code table we expect, for example, that the most similar codons - that is, those that differ by only one base - will have more similar physicochemical properties. Therefore, the 64 codons of the genetic code table ordered in a certain way would also represent an ordering of some of their physicochemical properties. Now, a study aimed at clarifying which physicochemical property of amino acids has influenced the allocation of amino acids in the genetic code has established that the partition energy of amino acids has played a role decisive in this. Indeed, under some conditions, the genetic code was found to be approximately 98% optimized on its columns. In this same work, it was shown that this was most likely the result of the action of natural selection. If natural selection had truly allocated the amino acids in the genetic code in such a way that similar amino acids also have similar codons - this, not through a mechanism of physicochemical interaction between, for example, codons and amino acids - then it might turn out that even different physicochemical properties of codons (or anticodons or bases) show some correlation with the physicochemical properties of amino acids, simply because the partition energy of amino acids is correlated with other physicochemical properties of amino acids. It is very likely that this would inevitably lead to a correlation between codons (or anticodons or bases) and amino acids. In other words, since the codons (anticodons or bases) are ordered in the genetic code, that is to say, some of their physicochemical properties should also be ordered by a similar order, and given that the amino acids would also appear to have been ordered in the genetic code by selection natural, then it should inevitably turn out that there is a correlation between, for example, the hydrophobicity of anticodons and that of amino acids. Instead, the intervention of natural selection in organizing the genetic code would appear to be highly compatible with the main mechanism of structuring the genetic code as supported by the coevolution theory. This would make the coevolution theory the only plausible explanation for the origin of the genetic code.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

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.

Ribosomal incorporation of β-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 β-amino acid incorporation. However, multiple/consecutive incorporations of β-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of β-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of β-homophenylglycine (βPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive βPhg\'s. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for β-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 β-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 β-amino acids.