PLoS Biol

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features

Insights into the Co-Evolution of Ribosomal Protein S15 with its Regulatory RNAs

Thesis advisor: Michelle M. MeyerRibosomes play a vital role in all cellular life translating the genetic code into functional proteins. This pivotal function is derived from its structure. The large and small subunits of the ribosome consist of 3 ribosomal RNA strands and over 50 individual ribosomal proteins that come together in a highly coordinated manner. There are striking differences between eukaryotic and prokaryotic ribosomes and many of the most potent antibacterial drugs target bacterial ribosomes (e.g. tetracycline and kanamycin). Bacteria spend a large amount of energy and nutrients on the production and maintenance of these molecular machines: during exponential growth as much as 40% of dry bacterial mass is ribosomes (Harvey 1970). Because of this, bacteria have evolved an elegant negative feedback mechanism for the regulation of their ribosomal proteins, known as autoregulation. When excess ribosomal protein is produced, unneeded for ribosome assembly, the protein binds a structured portion of its own mRNA transcript to prevent further expression of that operon. Autoregulation facilitates a quick response to changing environmental conditions and ensures economical use of nutrients. My thesis has investigated the autoregulatory function of ribosomal protein S15 in diverse bacterial phyla. In many bacterial species, when there is excess S15 the protein interacts with an RNA structure formed in the 5’-UTR of its own mRNA transcript that enables autoregulation of the S15-encoding operon, rpsO. For many ribosomal proteins (ex. L1, L20, S2) there is striking homology and often mimicry between the recognition motifs within the rRNA and the regulatory mRNA structure. However, this is not the case for S15-three different regulatory RNA structures have been previously described in E. coli, G. stearothermophilus, and T. thermophilus (Portier 1990, Scott 2001, Serganov 2003). These RNAs share little to no structural homology to one another, nor the rRNA, and they are narrowly distributed to their respective bacterial phyla, Gammaproteobacteria, Firmicutes, and Thermales. It is unknown which regulatory RNA structures control the expression of S15 outside of these phyla. Additionally, previous work has shown the S15 homolog from G. stearothermophilus is unable to regulate expression using the mRNA from E. coli. These observations formulate the crux of the question this thesis work endeavors to answer: What drove the evolution of such diverse regulatory RNA structures in these different bacteria? In Chapter II, “Discover and Validate Novel Regulatory Structures for Ribosomal Protein S15in Diverse Bacterial Phyla”, I present evidence for the in silico identification of three novel regulatory RNA structures for S15 and present experimental evidence that one of these novel structures is distinct from those previously described. In Chapter III, “Co-evolution of Ribosomal Protein S15 with Diverse Regulatory RNA Structures”, I present evidence that the amino acid differences in S15 homologs contribute to differences in mRNA binding profiles, and likely lead to the development of the structurally diverse array of the regulatory RNAs we observe in diverse bacterial phyla. In Chapter IV, “Synthetic cis-regulatory RNAs for Ribosomal Protein S15”, I investigate the derivation of novel cis-regulatory RNAs for S15 and find novel structures are readily-derived, yet interact with the rRNA-binding face of S15. Together the work presented in this thesis advances our understanding of the co-evolution between ribosomal protein S15 and its regulatory RNAs in diverse bacterial phyla.Thesis (PhD) — Boston College, 2016.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Development and Evaluation of a Fluorescent Activated Droplet Sorting Regulatory Assay for Ribosomal Cis-Regulatory RNAs:

Thesis advisor: Michelle M. MeyerExisting methods of assaying the function of cis-regulatory RNAs come with significant drawbacks when assaying large RNA libraries. Highly sensitive cell-based assays such as the β galactosidase assay are labor intensive, difficult to scale up and may lose sensitivity with increased throughput. GFP and luciferase reporters can be used with FACS to increase assay throughput, but sorting small bacterial cells is challenging and greatly reduces assay sensitivity. Conversely, in vitro methods allow for fast screening of very large RNA libraries, but only select for properties of binding, not regulation. By combining the principles of classic in cell regulatory assays with modern tools, cis-regulatory RNAs can be quickly screened for regulatory activity at a large scale. The assay under development, Fluorescent Activated Droplet Sorting Regulatory Assay (FADSRA), uses microfluidics to encapsulate single cells expressing a fluorescent protein under the control of a cis-regulatory RNA. These cells are then cultured into microcolonies within the droplets, which are subsequently sorted according to fluorescent signal. Deep amplicon sequencing of the regulatory RNAs can then reveal which sequences can regulate and which cannot. Thus, FADSRA can help bridge the gap between in vitro RNA binding and gene regulation assays, providing a way to answer sophisticated questions about cis-regulatory RNAs requiring high-throughput assay methods. While many applications for FADSRA are possible, such as verifying regulatory activity of in vitro binders or screening synthetic regulators, one such application of FASDRA is the creation of fitness landscapes that probe sequence-function relationships of RNA cis-regulators. This dissertation first develops and optimizes the regulatory assay for ribosomal leaders in Chapter 2, following by creating a single mutant fitness landscape of the E. coli S15 leader RNA in Chapter 3. Results of this fitness landscape largely support previously published mutational studies and highlight the necessity of stable hairpin formation for regulation of the E. coli S15 leader. Chapter 4, examining the regulation of S15 protein and leader homologs, and Chapter 5, testing the adaptability of FADSRA to other cis-regulatory RNAs, examine possible further applications of the assay.Thesis (PhD) — Boston College, 2022.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Cold shock response of Salmonella enterica serovar typhimurium; the involvement of the CspA paralogues

Salmonella enterica sv. typhimurium is a major food-borne pathogen, in part because of its ability to persist and multiply at low temperatures. Adaptation to refrigerated temperatures involves induction of a multigenic cold shock response (CSR); where gene expression is co-ordinately modified, to express cold shock proteins (CSPs). Characterisation of CspA, the major cold shock protein, instigated the identification of other CspA paralogues; which are highly conserved and widespread across species. Six CspA paralogues have previously been identified in S. typhimurium and a csp null strain, lacking all CspA paralogues made. This strain is unable to grow following cold shock, demonstrating that the CspA paralogues play an essential role during low temperature adaptation. The individual CspA paralogues exhibit distinct expression profiles; including expression of CspC and CspE at optimal temperature and CspA and CspB following cold shock. This work investigates the transcriptional changes of S. typhimurium during cold shock and the role of the CspA paralogues under both optimal and cold shock conditions. Using a bacteriophage Mu transposon library (Francis and Gallagher, 1993) this study identifies 7 novel cold induced targets and analyses their native expression levels in SL1344 and the csp null strain during cold shock. This revealed that the regulation of 5 discrete loci including tRNApro2, cpxP and 3 uncharacterised ORFS are mediated by CspA paralogues. In addition, the transcriptional profiles of a highly conserved and essential set of genes encoding known cold shock proteins, NusA, IF2, RbfA, PNPase and CsdA have been characterised. Comparative Northern analysis of SL1344 and the csp null strain has identified a role for CspA paralogues in mediating low temperature induction of three of these genes, through transcription anti-termination. Taken together these results demonstrate that during adaptation to low temperature CspA paralogues regulate expression of genes involved in the translational machinery and metabolic biosynthetic pathways: possibly through a number of transcriptional and post transcriptional processing events. Furthermore this study provides in vivo evidence of the RNA binding activity of the S. typhimurium CspA paralogues. Using fusion proteins, the RNA targets of CspE at 37°C and CspA at 10°C were isolated and analysed. This work identifies 17 direct binding targets for CspE and these indicate that CspE performs a role at optimal growth temperature in regulating components of metabolic (coaA and plsX), translational (EF-Tu, EF-G and IF3) and virulence associated (hha) pathways. Functional redundancy between CspE and CspA was suggested as both paralogues bound 16s rRNA. In light of these findings, the functions of CspA & CspE at optimal and low temperature are discussed. Overall this study has revealed novel information about low temperature adaptation of S. typhimurium, expanding our knowledge of the complexity and importance of the CSR in bacterial pathogens. In addition this work enhances our comprehension of the roles of the CspA paralogues at both optimal and low temperature

Etude de la maturation cytoplasmique de la petite sous-unité ribosomique chez Saccharomyces cerevisiae

Les ribosomes constituent un des acteurs majeurs du mécanisme de traduction dans toute cellule vivante. La synthèse des ribosomes est un processus complexe commençant par la transcription d'un pré-ARN ribosomique (ARNr) contenant les futurs ARNr matures ainsi que des séquences qui vont être coupées tout au long de la biogenèse des sous-unités ribosomiques. Chez Saccharomyces cerevisiae, ce ne sont pas moins de 200 facteurs qui interviennent tout au long de ce processus. Nous nous sommes intéressés plus précisément à l'étape cytoplasmique de maturation de la petite sous-unité ribosomique, consistant en une coupure endonucléolytique permettant de passer d'un pré-ARNr 20S contenu dans une particule pré-40S à un ARNr 18S mature contenu dans une sous-unité 40S. Cette sous-unité mature peut ensuite initier la traduction. Le modèle initial proposait que la maturation de la petite sous-unité fût un prérequis à l'initiation de la traduction. Nos expériences ont permis d'observer qu'une fraction du pré-ARNr 20S cosédimente avec les complexes de 80S et polysomes. Cette fraction de pré-ARNr 20S est augmentée dans certains mutants où l'on bloque la maturation de la petite-sous unité dans le cytoplasme. Nous avons confirmé l'existence de ribosomes contenant des particules pré-40S et interagissant avec des ARNm par des approches biochimiques. Ainsi, nos données suggèrent que des sous-unités ribosomiques non matures peuvent initier la traduction. Ces ribosomes aberrants sont alors dégradés via le No Go decay, un mécanisme de contrôle-qualité des ARNs cytoplasmiques. Ainsi, le No Go Decay fonctionnerait comme le mécanisme ultime de contrôle-qualité des sous-unités ribosomiques 40S.Ribosomes constitute one of the major actors of the mechanism of translation in any living cell. The synthesis of ribosomes is a complex process beginning with the transcription of a pre-ribosomal RNA (rRNA) containing future mature rRNAs as well as sequences that are eliminated during ribosome biogenesis. In Saccharomyces cerevisiae, no less than 200 factors are implicated in this process. We were more precisely interested in the cytoplasmic step of the small ribosomal subunit maturation consisting of an endonucleolytic cleavage of the 20S pre-rRNA contained in a pre-40S particle and leading to the mature 18S rRNA contained in the 40S ribosomal subunit. The initial model was that 40S ribosomal subunit maturation might be a pre-requisite for translation initiation. Our experiments have led to the observation that a fraction of 20S pre-rRNA co-sediments with 80S complexes and polysomes. This 20S pre-rRNA fraction can be increased in mutants impaired in the cytoplasmic step of 40S ribosomal subunit maturation. By biochemical approaches, we confirmed the occurrence of ribosomes containing pre-40S particles and mRNAs. Thus, our data suggest that pre-40S particles can initiate translation. These aberrant ribosomes are then degraded via the No Go decay pathway involved in the quality control of some cytoplasmic RNAs. No-Go Decay would function as an ultimate quality control mechanism of the 40S ribosomal subunit

Combinatorial stress response of the fungal pathogen Candida glabrata

Candida glabrata is an opportunistic human fungal pathogen, with an increasing incidence of infection, as well as an innate resistance to antifungal drug therapies. It is more closely related to the model and non-pathogenic yeast, Saccharomyces cerevisiae, than other Candida spp. Previous studies have only focused on the response to independent stressors therefore little is known about the adaptive response to simultaneous stresses, even though this is likely to be more relevant in an ecological and pathophysiological setting e.g. upon macrophage engulfment. This study was conducted with the hypothesis that the response of C. glabrata to stressors applied simultaneously could not be explained by simply combining the response to single stresses. To investigate this hypothesis, the response of C. glabrata to hyperosmotic and oxidative stressors applied singly and in combination were examined by timecourse microarray analysis and functional genomics. While genes involved in a HOG-like (High Osmolarity Glycerol) response were regulated by C. glabrata under hyperosmotic stress, many homologous genes are not observed to be regulated by S. cerevisiae. The phenotypes displayed by null mutants of the HOG pathway implicate this MAPK signalling pathway in not only hyperosmotic stress, but also cell wall integrity and metal ion resistance. Microarray analysis revealed a prolonged transcriptional regulation over time with increasing concentration of oxidative stress and other genes with a similar pattern of expression were identified and studied. Transcript profiling of a strain lacking the key oxidative stress regulator Yap1, along with bioinformatic analysis of its binding sites, identified possible targets of this transcription factor in C. glabrata under oxidative stress. This study has identified differentially regulated transcript profiles unique to simultaneous stress and not seen under single stress conditions, indicating that a specific transcriptional response is required for C. glabrata to respond and adapt to combinatorial stress; it is not simply the addition of two individual responses. Comparisons of the transcriptional analysis presented here with that of published macrophage engulfed C. glabrata cells revealed that combinatorial stress elicits a similar response as the host environment. Combining functional genomics and transcript profiling under stress has allowed the identification and characterisation of genes involved in stress response as well as the construction of diagrams specific to the response of C. glabrata to stress

Circadian clock orchestration of signaling pathways influences mouse metabolism

Circadian clocks, present in organisms leaving in a rhythmic environment, constitute the mechanisms allowing anticipation and adaptation of behavior and physiology in response to these environmental variations. As a consequence, most aspects of metabolism and behavior are under the control of this circadian clock. At a molecular level, in all the studied species, the rhythmic expression of the genes involved are generated by interconnected transcriptional and translational feedback loops. In mammals, the heterodimer composed of BMAL1 and its partners CLOCK or NPAS2 constitutes a transcriptional activator regulating transcription of Per and Cry genes. These genes encode for repressors of the activity of BMAL1:CLOCK or BMAL1: NPAS2 heterodimers, thus closing a negative feedback loop that generates rhythms of approximately 24 hours. The aim of my doctoral work consisted in the investigation of the role of circadian clock in the regulation of different aspects of mouse metabolism through the rhythmic activation of signaling pathways. First, we showed that one way how the circadian clock exerts its function as an oscillator is through the regulation of mRNA translation. Indeed, we present evidence showing that circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. In the second part, we showed the involvement of the circadian clock in lipid metabolism. Indeed, the three PAR bZip transcription factors DBP, TEF and HLF, are regulated by the molecular clock and play key roles in the control of lipid metabolism. Here we present evidence concerning the circadian expression and activity of PPARα via the circadian transcription of genes involved in the release of fatty acids, natural ligands of PPARα. It leads to the rhythmic activation of PPARα itself which could then play its role in the transcription of genes encoding proteins involved in lipid, cholesterol and glucose metabolism. In addition, we considered the possible role of lipid transporters, here SCP2, in the modulation of circadian activation of signaling pathways such as TORC1, PPARα and SREBP, linked to metabolism, and its feedback on the circadian clock. In the last part of this work, we studied the effects of these circadian clock-orchestrated pathways in physiology, as clock disruptions have been shown to be linked to metabolic disorders. We performed in vivo experiments on genetically and high-fat induced obese mice devoid of functional circadian clock. The results obtained showed that clock disruption leads to impaired triglycerides and glucose homeostasis in addition to insulin secretion and sensitivity. -- Les rythmes circadiens, présents chez tout organisme vivant dans un environnement rythmique, constituent l'ensemble de mécanismes permettant des réponses comportementales et physiologiques anticipées et adaptées aux variations environnementales. De ce fait, la plupart des aspects liés au métabolisme et au comportement de ces organismes apparaissent être sous le contrôle de l'horloge circadienne contrôlant ces rythmes. Au niveau moléculaire, dans toutes les espèces étudiées, l'expression rythmique de gènes impliqués sont générés par l'interconnexion de boucles de contrôle transcriptionnelles et traductionnelles. Chez les mammifères, l'hétérodimère composé de BMAL1 et de ses partenaires CLOCK ou NPAS2 constitue un activateur transcriptionnel régulant la transcription des gènes Per et Cry. Ces gènes codent pour des répresseurs de l'activité des hétérodimères BMAL1:CLOCK ou BMAL1:NPAS2. Cela a pour effet de fermer la boucle négative, générant ainsi des rythmes d'environ 24 heures. Le but de mon travail de thèse a consisté en l'investigation du rôle de l'horloge circadienne dans la régulation de certains aspects du métabolisme chez la souris via la régulation de l'activation rythmique des voies de signalisation. Nous avons tout d'abord montré que l'horloge circadienne exerce sa fonction d'oscillateur notamment au niveau de la régulation de la traduction des ARNm. En effet, nous présentons des preuves montrant que l'horloge circadienne influence la traduction temporelle d'un groupe d'ARNm impliqués dans la biogénèse des ribosomes en contrôlant la transcription de facteurs d'initiation de la traduction ainsi que l'activation rythmique des voies de signalisation qui sont impliquées dans leur régulation. De plus, l'oscillateur circadien régule la transcription d'ARNm codant pour les protéines ribosomales et d'ARN ribosomaux. De cette façon, l'horloge circadienne exerce un rôle majeur dans la coordination des étapes de transcription et traduction permettant la biogénèse des ribosomes. Dans la deuxième partie, nous montrons les implications de l'horloge circadienne dans le métabolisme des lipides. En effet, DBP, TEF et HLF, trois facteurs de transcription de la famille des PAR bZip qui sont régulés par l'horloge circadienne, jouent un rôle clé dans le contrôle du métabolisme des lipides par l'horloge circadienne. Nous apportons ici des preuves concernant l'expression et l'activité rythmiques de PPARα via la transcription circadienne de gènes impliqués dans le relargage d'acides gras, ligands naturels de PPARα, conduisant à l'activation circadienne de PPARα lui-même, pouvant ainsi jouer son rôle de facteur de transcription de gènes codant pour des protéines impliquées dans le métabolisme des lipides, du cholestérol et du glucose. De plus, nous nous sommes penchés sur le rôle possible de transporteurs de lipides, ici SCP2, dans la modulation de l'activation circadienne de voies de signalisation, telles que TORC1, PPARα et SREBP, qui sont liées au métabolisme, ainsi que son impact sur l'horloge elle-même. Dans la dernière partie de ce travail, nous avons étudié les effets de l'activation de ces voies de signalisation régulées par l'horloge circadienne dans le contexte physiologique puisqu'il a été montré que la perturbation de l'horloge pouvait être associée à des désordres métaboliques. Pour ce faire, nous avons fait des expériences in vivo sur des souris déficientes pour l'horloge moléculaire pour lesquelles l'obésité est induite génétiquement ou induite par la nourriture riche en lipides. Les résultats que nous obtenons montrent des dérèglements au niveau de l'homéostasie des triglycérides et du glucose ainsi que sur l'expression et la réponse à l'insuline