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.

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.

Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP.

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

eIF3 Associates with 80S Ribosomes to Promote Translation Elongation, Mitochondrial Homeostasis, and Muscle Health.

eIF3, a multi-subunit complex with numerous functions in canonical translation initiation, is known to interact with 40S and 60S ribosomal proteins and translation elongation factors, but a direct involvement in translation elongation has never been demonstrated. We found that eIF3 deficiency reduced early ribosomal elongation speed between codons 25 and 75 on a set of ∼2,700 mRNAs encoding proteins associated with mitochondrial and membrane functions, resulting in defective synthesis of their encoded proteins. To promote elongation, eIF3 interacts with 80S ribosomes translating the first ∼60 codons and serves to recruit protein quality-control factors, functions required for normal mitochondrial physiology. Accordingly, eIF3e+/- mice accumulate defective mitochondria in skeletal muscle and show a progressive decline in muscle strength. Hence, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for mitochondrial physiology and muscle health.

tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa.

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 Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.

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.

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. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

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. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease-causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain-domain interactions that are otherwise perturbed by each individual mutation.

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. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.

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. We extended the classical 'induced-fit' model of initial selection by incorporating wb-WC reaction parameters in open and closed states. For predicted parameters, the non-equilibrium exoergic wb-WC reaction is kinetically limited by the decoding rates. The model explains early observations of the WC geometry of G•U from equilibrium structural studies and reveals discrimination capacity for the working ribosome operating at non-equilibrium conditions. The equilibration of the exoergic wb-WC reaction counteracts the equilibration of the open-closed transition of the A site, constraining the decoding accuracy and potentially explaining the persistence of the G•U as an error hotspot. Our results unify structural and mechanistic views of codon-anticodon decoding and generalize the 'induced-fit' model for flexible substrates.

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.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.

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. Multiple identifiers of ORFs that use the 3-nt periodicity in Ribo-Seq data sets have been successful in eukaryotic smORF annotation. They have difficulties evaluating prokaryotic genomes due to the unique architecture (e.g. polycistronic messages, overlapping ORFs, leaderless translation, non-canonical initiation etc.). Here, we present a new algorithm, smORFer, which performs with high accuracy in prokaryotic organisms in detecting putative smORFs. The unique feature of smORFer is that it uses an integrated approach and considers structural features of the genetic sequence along with in-frame translation and uses Fourier transform to convert these parameters into a measurable score to faithfully select smORFs. The algorithm is executed in a modular way, and dependent on the data available for a particular organism, different modules can be selected for smORF search.

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. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.

The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.

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. However, after adjusting for other factors, e.g. ECOG performance status and patients' age, this survival advantage vanished. Our analysis suggests that systemic pelvic and para-aortic lymphadenectomy was not associated with an improvement of the progression-free and overall survival of patients with optimally debulked ovarian cancer.

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 m6A-modified mRNAs recovered for translation compared to unmodified mRNAs, i.e. 95% vs 84%, respectively. Considering structural mRNA analysis, we found that the m6A modification enhances structuring at nucleotides in its close vicinity. Our results suggest that SG-sequestered mRNAs disassemble nearly completely from SGs and the m6A modification may display some advantage to the mRNAs in their recovery for translation likely by m6A-driven structural stabilization.

Invariable stoichiometry of ribosomal proteins in mouse brain tissues with aging.

Across phyla, the ribosomes-the central molecular machines for translation of genetic information-exhibit an overall preserved architecture and a conserved functional core. The natural heterogeneity of the ribosome periodically phases a debate on their functional specialization and the tissue-specific variations of the ribosomal protein (RP) pool. Using sensitive differential proteomics, we performed a thorough quantitative inventory of the protein composition of ribosomes from 3 different mouse brain tissues, i.e., hippocampus, cortex, and cerebellum, across various ages, i.e., juvenile, adult, and middle-aged mouse groups. In all 3 brain tissues, in both monosomal and polysomal ribosome fractions, we detected an invariant set of 72 of 79 core RPs, RACK1 and 2 of the 8 RP paralogs, the stoichiometry of which remained constant across different ages. The amount of a few RPs punctually varied in either one tissue or one age group, but these fluctuations were within the tight bounds of the measurement noise. Further comparison with the ribosomes from a high-metabolic-rate organ, e.g., the liver, revealed protein composition identical to that of the ribosomes from the 3 brain tissues. Together, our data show an invariant protein composition of ribosomes from 4 tissues across different ages of mice and support the idea that functional heterogeneity may arise from factors other than simply ribosomal protein stoichiometry.

Polymerase III transcription is necessary for T cell priming by dendritic cells.

Exposure to microbe-associated molecular patterns (MAMPs) causes dendritic cells (DCs) to undergo a remarkable activation process characterized by changes in key biochemical mechanisms. These enhance antigen processing and presentation, as well as strengthen DC capacity to stimulate naïve T cell proliferation. Here, we show that in response to the MAMPS lipopolysaccharide and polyriboinosinic:polyribocytidylic acid (Poly I:C), RNA polymerase III (Pol lII)-dependent transcription and consequently tRNA gene expression are strongly induced in DCs. This is in part caused by the phosphorylation and nuclear export of MAF1 homolog negative regulator of Poll III (MAF1), via a synergistic casein kinase 2 (CK2)- and mammalian target of rapamycin-dependent signaling cascade downstream of Toll-like receptors (TLRs). De novo tRNA expression is necessary to augment protein synthesis and compensate for tRNA degradation driven by TLR-dependent DC exposure to type-I IFN. Although protein synthesis is not strongly inhibited in absence of RNA Pol III activity, it compromises the translation of key DC mRNAs, like those coding for costimulatory molecules and proinflammatory cytokines, which instead can be stored in stress granules, as shown for CD86 mRNA. TLR-dependent CK2 stimulation and subsequent RNA Pol III activation are therefore key for the acquisition by DCs of their unique T cell immune-stimulatory functions.

Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.

tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies.

A rare heterozygous TREM2 coding variant identified in familial clustering of dementia affects an intrinsically disordered protein region and function of TREM2.

Rare coding variants in the triggering receptor expressed on myeloid cells-2 (TREM2) gene have been associated with Alzheimer disease (AD) and homozygous TREM2 loss-of-function variants have been reported in families with monogenic frontotemporal-like dementia with/without bone abnormalities. In a whole-exome sequencing study of a family with probable AD-type dementia without pathogenic variants in known autosomal dominant dementia disease genes and negative for the apolipoprotein E (APOE) ε4 allele, we identified an extremely rare TREM2 coding variant, that is, a glycine-to-tryptophan substitution at amino acid position 145 (NM_018965.3:c.433G>T/p.[Gly145Trp]). This alteration is found in only 1 of 251,150 control alleles in gnomAD. It was present in both severely affected as well as in another putatively affected and one 61 years old as yet unaffected family member suggesting incomplete penetrance and/or a variable age of onset. Gly145 maps to an intrinsically disordered region (IDR) of TREM2 between the immunoglobulin-like and transmembrane domain. Subsequent cellular studies showed that the variant led to IDR shortening and structural changes of the mutant protein resulting in an impairment of cellular responses upon receptor activation. Our results, suggest that a p.(Gly145Trp)-induced structural disturbance and functional impairment of TREM2 may contribute to the pathogenesis of an AD-like form of dementia.