Translational quality control mechanisms that mitigate stop codon readthrough and ensure protein homeostasis

Cells invest tremendously to maintain the fidelity for transcription and translation to ensure accurate transmission of the genetic code into proteins. Yet, errors may occur at each stage. During transcription, errors arise at a rate of ~10-5-10-4 per base, whereas the error rate during translation is around a magnitude higher with ~10-4-10-3 amino acid misincorporations per codon. Such errors become increasingly frequent with ageing, posing a sizable risk for the organism. In some instances, this leads to missing or misread stop codons, allowing translation to continue into the 3’UTRs of transcripts. Such C-terminal extensions may interfere with the folding of proteins, or worse, promote promiscuous interactions with other proteins, which in turn may disturb cellular processes and reduce overall fitness. Translation into the polyA-tail of transcripts leads to the activation of the ribosome quality control (RQC) complex, which clears both aberrant protein and mRNA. However, in most cases, translation would be terminated at stop codons within the 3’UTR before the ribosome reaches the polyA-tail. Such readthrough events would therefore not be recognized by the RQC. While previous studies suggested that such readthrough products are recognized and efficiently cleared by cells, the underlying mechanism remained unclear. Given the decline in translational fidelity during ageing, this clearance pathway is expected to become increasingly important to release the burden on the proteostasis network. Using the nematode C. elegans as a model for ageing, we aimed to identify the quality control mechanisms mitigating translational readthrough and investigated the consequences of their failure during ageing. Using this approach, we identified in C. elegans and human cells that readthrough proteins are cleared through a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay mediated by GCN1 and CCR4/NOT limits the accumulation of readthrough proteins. Selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter non-optimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly affecting transmembrane proteins and collagens during ageing. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis

Statistical physics approaches to protein translation

In this work we present an integrated approach to the study of protein translation, based on Statistical Physics. We adopted three different but complementary perspectives: building hypothesis up from the data, modeling down from reasonable assumptions, and using computer simulations when everything else fails. In particular, we first analyze the mRNA sequences by means of inforation theory. We focus on the way the redundancy of the genetic code (the 61 sense triplets of nucleotides -the codons- encode for 20 amino acids) is utilized in the actual sequences, a phenomenon known as the codon bias. We observe that it is not completely random, and encodes information in the frequencies and in the order of the codons. With the scope of explaining these anomalies, we develop and analyze a family of stochastic models. Translation emerges as a systemic process, where the limited amount of resources in the cell couples the expression of the genes at a global level. We also suggest a game-theoretical interpretation of the codon bias. We finally attack the problem of cotranslational folding (i.e., the folding of the protein while it is still being translated). Specifically, we ask how this process depends on the order of the codons. This question is computationally very cumbersome. We thus propose a framework based on Markov chains, which allows the efficient simulation of arbitrarily complicate cotranslational folding mechanisms

Protein folding on the ribosome studied using NMR spectroscopy

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome-nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity

Ribosomal History Reveals Origins of Modern Protein Synthesis

The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world

rpsA and ribosomal protein S1: investigating a non-canonical translation initiation element

Translation initiation rates are fine-tuned by altering interactions between the ribosome and the translation initiation region of an mRNA. In bacteria varying levels of RNA structure attenuate these interactions by masking the ribosome-binding site from the small ribosomal subunit. Recent studies have described diverse strategies for recruiting mRNA to the ribosome, and highlighted the contributions of ribosomal protein S1. Here, I provide evidence that the non-canonical initiation mechanism that governs translation of the rpsA mRNA, encoding ribosomal protein S1, contains a three-dimensional architecture that is required for efficient translation. Furthermore, S1 plays an essential role during the initiation phase of translation by recruiting mRNA to the ribosome—unfolding structured mRNAs, and allowing for correct start codon positioning on the ribosome. Combining crosslinking immunoprecipitation and high-throughput sequencing approaches revealed the extent of S1’s involvement in mRNA recruitment, while also highlighting a broader role as a regulator of many RNA classes.Alberta Innovates (Strategic Chairs Program) [SC60-T2

Neue Perspektiven auf post-transkriptionale Regulationsmechanismen in Pseudomonas aeruginosa

Regulation of gene expression plays a key role in bacterial adaptability to changes in the environment. Quorum sensing (QS) systems constitute an integral part of this gene regulatory network and coordinate bacterial responses under high cellular densities. In the nosocomial pathogen Pseudomonas aeruginosa, a sophisticated QS network exists controlling virulence factor synthesis important for pathogenesis. The overall aim of this thesis was to gain insight into novel regulation mechanisms of QS-based gene expression at the post-transcriptional level. In this context, we characterized the S-adenosyl-L-methionine-dependent methyltransferase (PrmC), specifically methylating a conserved GGQ motif of peptide chain release factors. Mutation of prmC was shown to predominantly affect the expression of major virulence-associated factors and caused attenuation of virulence in a Galleria mellonella larvae infection model. Furthermore, a previously unrecognized post-transcriptional regulation mechanism in bacteria was characterized. This involved an RhlR-mediated transcriptional induction of a secondary structure in the 5' untranslated region of the pqs operon transcript. The resulting hairpin-like structure sterically hinders ribosomal access to the ribosome binding site thus repressing pqs signaling. Another important focus of the work was to establish ribosome profiling (Ribo-Seq). The technique Ribo-Seq is based on deep sequencing of ribosome-protected mRNA fragments and provides a global snapshot of translation in vivo. This powerful tool opens up wide-ranging prospects to the developing picture of post-transcriptional regulation in P. aeruginosa. Finally, the present thesis focuses on the role of PqsE as part of the pqs QS system in regulating QS-dependent virulence factor production. We provide evidence that PqsE is an enzyme that interferes with carbon flow at the chorismate branching point in the complex metabolic network of P. aeruginosa. By using a multi-methodological approach, we suggest that PqsE is involved in the biosynthesis of phenylpyruvate, being a possible signal molecule for the rhl QS system.Regulation der Genexpression spielt eine zentrale Rolle in der bakteriellen Anpassungsfähigkeit an sich verändernde Umweltbedingungen. Quorum sensing (QS) Systeme sind ein wesentlicher Bestandteil dieses regulatorischen Netzwerkes und koordinieren bakterielle Reaktionen bei hoher Zelldichte. In dem nosokomialen Pathogen Pseudomonas aeruginosa existiert ein komplexes QS Netzwerk, welches die Synthese von Virulenzfaktoren kontrolliert die wichtig für die Pathogenese sind. Allgemeines Ziel dieser Dissertation war es, Einblicke in neuartige Regulationsmechanismen auf QS-basierender Genexpression auf dem post-transkriptionellen Level zu gewinnen. In diesem Zusammenhang haben wir die S-Adenosyl-L-Methionin-abhängige Methyltransferase (PrmC) charakterisiert, die spezifisch ein konserviertes GGQ Motif der Terminationsfaktoren methyliert. Es wurde gezeigt, dass prmC Mutation vorwiegend die Expression wichtiger Virulenz-assoziierter Faktoren betraf und eine Abschwächung der Virulenz in einem Galleria mellonella Larven Infektionsmodel zur Folge hatte. Zudem wurde ein zuvor unerkannter post-transkriptioneller Regulationsmechanismus charakterisiert. Dieser umfasst eine RhlR-vermittelte transkriptionelle Induktion einer Sekundärstruktur in der 5‘ nicht-codierenden Region des pqs Operon Transkripts. Die daraus resultierende Haarnadel-ähnliche Struktur verhindert sterisch den Zugang des Ribosoms an die ribosomale Bindungsstelle und unterdrückt auf diese Weise das pqs Signalsystem. Ein weiterer wichtiger Fokus dieser Arbeit bestand darin, ribosome profiling (Ribo-Seq) zu etablieren. Die Ribo-Seq Technik basiert auf dem deep sequencing von Ribosom geschützten mRNA Fragmenten, und verschafft eine globale Momentaufnahme der Translation in vivo. Dieses leistungsfähige methodische Werkzeug eröffnet umfangreiche Perspektiven im Hinblick auf das sich entwickelnde Bild der post-transkriptionellen Regulation in P. aeruginosa. Schließlich liegt ein Fokus dieser Dissertation auf der Rolle von PqsE als wichtiger Bestandteil des pqs QS Systems in der Regulation QS-abhängiger Virulenzfaktorproduktion. Wir konnten zeigen, dass PqsE ein Enzym ist, welches in den Kohlenstoffstrom an dem Chorisminsäure Verzweigungspunkt des komplexen metabolischen Netzwerkes von P. aeruginosa eingreift. Durch den Einsatz eines multi-methodischen Ansatzes schlagen wir vor, dass PqsE in der Phenylpyruvat Biosynthese involviert ist, welches ein mögliches Signalmolekül für das rhl QS System darstellt

翻訳調節と共発現遺伝子ネットワークに対する運動効果の網羅的解析

早大学位記番号:新7100早稲田大

The cumulative impact of chaperone mediated protein- folding during evolution

Molecular chaperones support protein folding and unfolding along with assembly and translocation of protein complexes. Chaperones have been recognized as important mediators between organismal genotype and phenotype as well as important maintainers of cellular fitness under environmental conditions that induce high mutational loads. This thesis presents recent studies revealing that the folding assistance supplied by chaperones is evident in genomic sequences, thus implicating chaperone-mediated folding as an influential factor during protein evolution. Furthermore the evolution and the symbiogenic origin of the eukaryotic chaperone repertoire are elucidated. Protein interaction with chaperones ensures a proper folding and function, yet an adaptation to obligatory dependence on such assistance may be irreversible, representing an evolutionary trap. Correlation between chaperone requirement and protein expression level indicate that the evolution of substrate-chaperone interaction is bounded by the required substrate abundance within the cell. Accumulating evidence suggests that the utility of chaperones is governed by a delicate balance between their help in mitigating the risks of protein misfolding and aggregate formation on the one hand, and the slower rate of protein maturation and the energetic cost of chaperone synthesis on the other

The rpsA gene and its product ribosomal protein S1: investigating the role of RNA structure in translation

The rpsA gene contains a translation initiation region (TIR) which lacks a Shine-Dalgarno element, is highly structured, and which efficiently drives translation in bacteria. The rpsA gene is downregulated by its gene product, ribosomal protein S1. To investigate how the structure of the TIR facilitates function both in translation and downregulation, we employed a combined SAXS, SHAPE, and computational approach. The structure-function relationship reveals that the introduction of single nucleotide substitutions in the TIR alters its structure, corresponding to a modulated translation efficiency in vivo. The activity of the rpsA TIR in a minimal in vitro transcription/translation system was investigated to understand the TIR’s requirement for additional factors. We observed that S1 is strictly required for its translation, and that S1 specifically recognizes and binds to the TIR when in excess over ribosomes. The structure-driven mechanism of this TIR may represent a previously overlooked strategy of translation initiation in bacteria.Alberta Innovates (Strategic Chairs Program [SC60-T2], Natural Sciences and Engineering Research Council of Canada Graduate Scholarship- Master