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.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen .

The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen .

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

The most abundant N-methyladenosine (mA) 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 mA remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single mA 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.

tRNA therapeutics for genetic diseases.

Transfer RNAs (tRNAs) have a crucial role in protein synthesis, and in recent years, their therapeutic potential for the treatment of genetic diseases - primarily those associated with a mutation altering mRNA translation - has gained significant attention. Engineering tRNAs to readthrough nonsense mutation-associated premature termination of mRNA translation can restore protein synthesis and function. In addition, supplementation of natural tRNAs can counteract effects of missense mutations in proteins crucial for tRNA biogenesis and function in translation.

Tailored Synthetic sRNAs Dynamically Tune Multilayer Genetic Circuits.

Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose rationally designed new small RNAs (sRNAs) that dynamically modulate gene expression of genetic circuits with a broad range (high, medium, and low) of repression. We designed multiple multilayer genetic circuits in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein.

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.

Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases. 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 translation.

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events.

Background: The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported.

Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.

In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E.

mRNAs sequestered in stress granules recover nearly completely for translation.

Stress granules (SGs) are membrane-less condensates composed of RNA and protein that assemble in response to stress stimuli and disassemble when stress is lifted. Both assembly and disassembly are tightly controlled processes, yet, it remains elusive whether mRNAs in SGs completely recover for translation following stress relief. Using RNA-seq of translating fractions in human cell line, we found that higher fraction of the mA-modified mRNAs recovered for translation compared to unmodified mRNAs, i.

Survival Advantage of Lymphadenectomy in Patients with Ovarian Cancer.

We investigated the survival effect of lymphadenectomy in ovarian cancer. The five-year progression-free and overall survival in early-stage ovarian cancer were not affected. Preliminary, unadjusted analysis in advanced ovarian cancer suggested an improvement in survival.

Immunoprecipation Assay to Quantify the Amount of tRNAs associated with Their Interacting Proteins in Tissue and Cell Culture.

Transfer RNAs (tRNAs) are highly abundant species and, along their biosynthetic and functional path, they establish interactions with a plethora of proteins. The high number of nucleobase modifications in tRNAs renders conventional RNA quantification approaches unsuitable to study protein-tRNA interactions and their associated functional roles in the cell. We present an immunoprecipitation-based approach to quantify tRNA bound to its interacting protein partner(s).

Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis.

The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the and transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in . We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient.

Solution Structure and Conformational Flexibility of a Polyketide Synthase Module.

Polyketide synthases (PKSs) are versatile C-C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties.

Constraints on error rate revealed by computational study of G•U tautomerization in translation.

In translation, G•U mismatch in codon-anticodon decoding is an error hotspot likely due to transition of G•U from wobble (wb) to Watson-Crick (WC) geometry, which is governed by keto/enol tautomerization (wb-WC reaction). Yet, effects of the ribosome on the wb-WC reaction and its implications for decoding mechanism remain unclear. Employing quantum-mechanical/molecular-mechanical umbrella sampling simulations using models of the ribosomal decoding site (A site) we determined that the wb-WC reaction is endoergic in the open, but weakly exoergic in the closed A-site state.

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNA but failed to release it, resulting in tRNA sequestration.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1.

RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system.

In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP.

smORFer: a modular algorithm to detect small ORFs in prokaryotes.

Emerging evidence places small proteins (≤50 amino acids) more centrally in physiological processes. Yet, their functional identification and the systematic genome annotation of their cognate small open-reading frames (smORFs) remains challenging both experimentally and computationally. Ribosome profiling or Ribo-Seq (that is a deep sequencing of ribosome-protected fragments) enables detecting of actively translated open-reading frames (ORFs) and empirical annotation of coding sequences (CDSs) using the in-register translation pattern that is characteristic for genuinely translating ribosomes.

Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens.

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaA and Listeria monocytogenes VgaL.

Towards the characterization of the hidden world of small proteins in Staphylococcus aureus, a proteogenomics approach.

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results.

Estimation of peptide elongation times from ribosome profiling spectra.

Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.

Codon Resolution Analysis of Ribosome Profiling Data.

Translation is a central biological process in living cells. Ribosome profiling approach enables assessing translation on a global, cell-wide level. Extracting versatile information from the ribosome profiling data usually requires specialized expertise for handling the sequencing data that is not available to the broad community of experimentalists.

Positive epistasis between disease-causing missense mutations and silent polymorphism with effect on mRNA translation velocity.

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome.