Translation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations.

Nonsense mutations - the underlying cause of approximately 11% of all genetic diseases - prematurely terminate protein synthesis by mutating a sense codon to a premature stop or termination codon (PTC). An emerging therapeutic strategy to suppress nonsense defects is to engineer sense-codon decoding tRNAs to readthrough and restore translation at PTCs. However, the readthrough efficiency of the engineered suppressor tRNAs (sup-tRNAs) largely varies in a tissue- and sequence context-dependent manner and has not yet yielded optimal clinical efficacy for many nonsense mutations. Here, we systematically analyze the suppression efficacy at various pathogenic nonsense mutations. We discover that the translation velocity of the sequence upstream of PTCs modulates the sup-tRNA readthrough efficacy. The PTCs most refractory to suppression are embedded in a sequence context translated with an abrupt reversal of the translation speed leading to ribosomal collisions. Moreover, modeling translation velocity using Ribo-seq data can accurately predict the suppression efficacy at PTCs. These results reveal previously unknown molecular signatures contributing to genotype-phenotype relationships and treatment-response heterogeneity, and provide the framework for the development of personalized tRNA-based gene therapies.

N6-methyladenosine in 5' UTR does not promote translation initiation.

The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.

Effect of mRNA/tRNA mutations on translation speed: Implications for human diseases.

Recent discoveries establish tRNAs as central regulators of mRNA translation dynamics, and therefore cotranslational folding and function of the encoded protein. The tRNA pool, whose composition and abundance change in a cell- and tissue-dependent manner, is the main factor which determines mRNA translation velocity. In this review, we discuss a group of pathogenic mutations, in the coding sequences of either protein-coding genes or in tRNA genes, that alter mRNA translation dynamics. We also summarize advances in tRNA biology that have uncovered how variations in tRNA levels on account of genetic mutations affect protein folding and function, and thereby contribute to phenotypic diversity in clinical manifestations.

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.

Teaching during the COVID-19 Pandemic: Sharing Results and Data Obtained from the Ames Test.

We present a resource for instructors that contains results and data sets from the Ames test. Our aim is to share the results we have collected in previous semesters with other instructors, so they will be able to "conduct" the Ames test without the need to set foot in a laboratory classroom. Instructors will be able to use our online resource to perform the test remotely, as a supplement to their laboratory classroom, or even under hybrid circumstances. The coronavirus disease 2019 (COVID-19) pandemic brought many changes, including the way we, as instructors, were able to carry out our educational curricula, since access to laboratory classrooms was not always possible. While COVID-19 restrictions are still in place, and thus access to laboratory classrooms is limited or null, instructors can use our online resource, without the need to set foot in a laboratory classroom. When COVID-19 restrictions are lifted and access to laboratory classrooms is permitted, instructors can follow the procedures we describe and compare their results with ours, which appear in Results and Discussion, or use our data sets as take-home assignments for their students. In addition to its use in detecting the potential mutagenicity of different samples, we have found the Ames test to be extremely useful for developing problem-solving skills by means of exercises like the ones included in this resource. Furthermore, the potential of this test as a starting point for problem-based learning is remarkable. Some suggestions for its use in active learning settings are provided.

On the Track of the Missing tRNA Genes: A Source of Non-Canonical Functions?

Cellular tRNAs appear today as a diverse population of informative macromolecules with conserved general elements ensuring essential common functions and different and distinctive features securing specific interactions and activities. Their differential expression and the variety of post-transcriptional modifications they are subject to, lead to the existence of complex repertoires of tRNA populations adjusted to defined cellular states. Despite the tRNA-coding genes redundancy in prokaryote and eukaryote genomes, it is surprising to note the absence of genes coding specific translational-active isoacceptors throughout the phylogeny. Through the analysis of different releases of tRNA databases, this review aims to provide a general summary about those "missing tRNA genes." This absence refers to both tRNAs that are not encoded in the genome, as well as others that show critical sequence variations that would prevent their activity as canonical translation adaptor molecules. Notably, while a group of genes are universally missing, others are absent in particular kingdoms. Functional information available allows to hypothesize that the exclusion of isodecoding molecules would be linked to: 1) reduce ambiguities of signals that define the specificity of the interactions in which the tRNAs are involved; 2) ensure the adaptation of the translational apparatus to the cellular state; 3) divert particular tRNA variants from ribosomal protein synthesis to other cellular functions. This leads to consider the "missing tRNA genes" as a source of putative non-canonical tRNA functions and to broaden the concept of adapter molecules in ribosomal-dependent protein synthesis.

Fine-tuning the metabolic rewiring and adaptation of translational machinery during an epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. METHODS: We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. RESULTS: Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already "Warburg-like" context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. CONCLUSIONS: Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.