A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets

Exosomal miRNA transfer is a mechanism for cell-cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip's amino-terminal domain, which was previously thought to mediate protein-protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip's RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5′ to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences. © 2018 The Author(s)

Single-particle cryo-electron microscopy studies of ribosomes with fragmented 28S rRNA

In the past five years, single-particle cryo-electron microscopy (cryo-EM) has revolutionized structural biology. Recent advances in detector technology and powerful computational methods now allow images of unprecedented detail to be recorded and structures to be determined at near-atomic resolution. For my PhD studies, I took advantage of this technique to study the structure of uniquely fragmented ribosomes. Ribosomes, are large macromolecular complexes that translate genetic information carried by messenger RNAs (mRNAs) into polypeptide chains. They are the protein production factories of all living cells and are thus involved in virtually all aspects of cellular development and maintenance. By virtue of their core role in the cell, ribosomes share a highly evolutionarily conserved core that carries out the fundamental processes of protein synthesis [1]. However, outside of this core, ribosome composition varies considerably. The main differences among eukaryotic ribosomes are due to rRNA expansion segments (ESs) and variations of ribosomal proteins (r-proteins) [1, 2]. Further, rRNA fragmentation occurring in regions of high variability has been reported in several organisms from bacteria to protozoa, insects, helminths, fish, and surprisingly mammals [3-15]. Recently, the naked mole-rat (Heterocephalus glaber) was discovered to have unusual cleavage sites in its 28S rRNA resulting in the deletion of the major part of the D6 variable region (ES15L) and leaving the two rRNA fragments disconnected [14]. The cleaved 28S rRNA has been associated with the naked mole rat’s increased translational fidelity [14]. The only other known mammals having fragmented rRNA are the tuco-tuco rodent (of the genus Ctenomys) and the degu (in the related genus Octodontomys) [13]. Here we present the high-resolution structures of the naked mole-rat, tuco-tuco, and guinea pig (Cavia porcellus) ribosomes. Guinea pig, which has canonical (non-fragmented) 28S rRNA is used as a rodent model for comparisons to the naked mole-rat and tuco-tuco ribosomes. During my PhD studies, I also looked at another uniquely fragmented ribosome, that of the protozoan parasite, Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease. The T. cruzi large subunit (LSU) rRNA is assembled from 8 pieces-5S, 5.8S, and six pieces forming jointly the 28S rRNA. Together with my colleagues from Joachim Frank’s and Liang Tong’s research groups, we solved the structure of the T. Cruzi LSU and identified distinctive trypanosome interactions, which allowed us to propose a tentative model for assembly of the large ribosomal subunit (60S) [16, 17]

EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb

A computational solution to automatically map metabolite libraries in the context of genome scale metabolic networks

This article describes a generic programmatic method for mapping chemical compound libraries on organism-specific metabolic networks from various databases (KEGG, BioCyc) and flat file formats (SBML and Matlab files). We show how this pipeline was successfully applied to decipher the coverage of chemical libraries set up by two metabolomics facilities MetaboHub (French National infrastructure for metabolomics and fluxomics) and Glasgow Polyomics (GP) on the metabolic networks available in the MetExplore web server. The present generic protocol is designed to formalize and reduce the volume of information transfer between the library and the network database. Matching of metabolites between libraries and metabolic networks is based on InChIs or InChIKeys and therefore requires that these identifiers are specified in both libraries and networks. In addition to providing covering statistics, this pipeline also allows the visualization of mapping results in the context of metabolic networks. In order to achieve this goal, we tackled issues on programmatic interaction between two servers, improvement of metabolite annotation in metabolic networks and automatic loading of a mapping in genome scale metabolic network analysis tool MetExplore. It is important to note that this mapping can also be performed on a single or a selection of organisms of interest and is thus not limited to large facilities

Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2 and substrate recruitment by the hexameric MecA–ClpC complex

Since the outbreak of the COVID-19 pandemic in December 2019, SARS-CoV-2 has generated awareness for the requirement of novel antiviral drugs that target new proteins. Previous studies have pointed to the pathogenic significance of the non-structural protein 1 (Nsp1), which was proposed to be a major virulence factor due to its dual role in host translation inhibition and viral replication of SARSCoV- 2. The precise mechanisms of these two functions of Nsp1 are still uncovered. Here, I report the backbone chemical-shift assignments and the atomic-resolution NMR structure of full-length Nsp1 from SARS-CoV-2 solved by NMR. I found that Cov-2 Nsp1 consists of a folded N-terminal domain and an unfolded C-terminal region. Previous studies have identified a surface of the folded N-terminal domain of Nsp1 that associates with both host mRNA, in its function as inhibitor of the host protein translation, and the viral 5’-UTR RNA, in its function as promoter of viral protein translation. I found that the acidic C-terminal tail of Nsp1 folds back on this surface and masks the RNA binding site. A recent Cryo-EM study has shown that the end of C-terminal region of Nsp1 interacts with the mRNA entry tunnel on the 40S subunit of the ribosome, thus inhibiting host mRNA entry and promoting its degradation. I propose that the RNA binding site on the Nsp1 N-terminal domain is protected by its C-terminal tail before Nsp1 contacts the ribosome. Upon ribosome binding, the C-terminal tail is displaced, and the RNA binding site is exposed to recruit either host mRNA or the viral 5’ UTR. My findings have consequences for the design of drugs targeting the RNA binding surface of Nsp1, as it demonstrates the need to develop a molecule that can not only recognize this surface with high affinity, but also displace the C-terminal tail to render this surface accessible. In bacteria, the unfoldase ClpC is a member of the conserved Hsp100/Clp family of AAA+ ATPases and is involved in various cellular processes. The functional form of ClpC is an hexameric assembly, which is responsible for controlled unfolding of substrate proteins. The ClpC hexamer can further associate with the protease ClpP to form a complete protein degradation machine. MecA functions as an adaptor protein of ClpC and is necessary to both promote the formation of the functional ClpC hexamer and to recruit specific substrate proteins, such as the transcription factor ComK. Degradation of ComK via MecA-mediated recruitment to the ClpCP complex is part of the regulatory mechanisms of the development of cell competence. The mechanisms of selective substrate recruitment are still unknown, due to the conspicuous conformational dynamics and heterogeneity that characterize this step. To understand how the substrate ComK is recruited to the MecA–ClpC complex, I reconstituted the ComK– MecA–ClpC complex in vitro and applied nuclear magnetic resonance (NMR) spectroscopy and other biophysical techniques, such as multi-angle light scattering. I found that addition of ComK stabilizes the MecA–ClpC complex by forming a homogeneous ternary protein complex, which contains a ClpC hexamer, four MecA and two ComK molecules in the presence of ATP. The structural differences between the MecA–ClpC and the ComK–MecA–ClpC complexes were also monitored by small-angle X-ray scattering datasets, which furthered confirmed the presence of the interaction between ComK and MecA. Seit dem Ausbruch der COVID-19-Pandemie im Dezember 2019 hat SARS-CoV-2 das Bewusstsein für den Bedarf an neuartigen antiviralen Medikamenten geweckt, die neue Proteine abzielen. Frühere Studien haben auf die pathogene Bedeutung des Unstrukturiertenproteins 1 (Nsp1) hingewiesen, das aufgrund seiner doppel Rolle bei der Inhibierung von Translationsprozessen im Wirt, sowie der viralen Replikation von SARSCoV- 2 als wichtiger Virulenzfaktor betrachtet wird. Die genauen Mechanismen dieser beiden Funktionen von Nsp1 sind noch weitesgehnd unerforscht. Ich mittels NMR erhobener Daten die Zuordnungen der chemischen Verschiebung des Rückgrats sowie die atomare NMR-Struktur von Nsp1 in voller Länge aus SARS-CoV-2. Ich fand heraus, dass Cov-2 Nsp1 aus einer gefalteten N-terminalen Domäne und einer ungefalteten C-terminalen Region besteht. Frühere Studien haben einen Oberflächenbereich der gefalteten N-terminalen Domäne von Nsp1 identifiziert, die sowohl mit Wirts-mRNA in ihrer Funktion als Inhibitor der Translation assoziiert, als auch mit der viralen 5'-UTR-RNA in ihrer Funktion als Promotor des viralen Proteins. Ich fand heraus, dass sich der saure C-terminale Emde von Nsp1 auf dieser Oberfläche zurückfaltet und die RNA-Bindungsstelle maskiert. Eine kürzlich durchgeführte Cryo-EM-Studie hat ergeben, dass das Ende der C-terminalen Region von Nsp1 mit dem mRNA-Eintrittstunnel auf der 40S-Untereinheit des Ribosoms interagiert, wodurch der mRNA-Eintritt des Wirts gehemmt und dessen Abbau gefördert wird. Ich schlage vor, dass die RNA-Bindungsstelle auf der N-terminalen Domäne von Nsp1 durch ihr C-terminalen Emde geschützt wird, bevor Nsp1 das Ribosom kontaktiert. Bei der Ribosomenbindung wird der C-terminale Bereich verschoben und die RNA-Bindungsstelle wird freigelegt, um entweder Wirts-mRNA oder die virale 5'-UTR zu rekrutieren. Meine Ergebnisse haben Konsequenzen für das Design von Medikamenten, die auf die RNA-bindende Oberfläche von Nsp1 abzielen, da sie die Notwendigkeit aufzeigen ein Molekül zu entwickeln, welches diese Oberfläche nicht nur mit hoher Affinität erkennen kann, sondern auch das flexible C-terminale Ende verdrängt, um diese Oberfläche zugänglich zu machen. In Bakterien ist die Unfoldase ClpC, ein Mitglied der Hsp100/Clp-Familie von AAA+ ATPasen, und ist an verschiedenen zellulären Prozessen beteiligt. Die funktionelle Form von ClpC folgt einer hexameren Anordnung, die für die kontrollierte Entfaltung von Substratproteinen verantwortlich ist. Das ClpC-Hexamer kann ferner mit der Protease ClpP assoziieren, um eine vollständige Proteinabbaumaschine zu bilden. MecA fungiert als Adapterprotein von ClpC und ist notwendig um sowohl die Bildung des funktionellen ClpCHexamers zu fördern, als auch spezifische Substratproteine wie unter anderem den Transkriptionsfaktor ComK zu rekrutieren. Der Abbau von ComK über MecA-vermittelte Rekrutierung zum ClpCP-Komplex ist Teil der Regulationsmechanismen der Entwicklung von Zellkompetenz. Dies liegt hauptsächlich an der auffällige Konformationsdynamik und Heterogenität des Komplexes, die diesen Schritt charakterisieren. Um zu verstehen, wie das Substrat ComK zum MecA-ClpC-Komplex rekrutiert wird, wurde der ComK-MecA-ClpCKomplex in vitro rekonstituiert und Kernspinresonanz(NMR)-Spektroskopie sowie weitere biophysikalische Techniken wie zum Beispiel Mehrwinkel-Lichtstreuung angewendet. Ich fand heraus, dass die Zugabe von ComK den MecA-ClpC-Komplex durch Bildung eines homogenen ternären Proteinkomplexes stabilisiert, der in Gegenwart von ATP ein ClpC-Hexamer, vier MecA- und zwei ComK-Moleküle enthält. Die strukturellen Unterschiede zwischen den MecA-ClpC- und den ComK-MecA-ClpC-Komplexen wurden auch durch Kleinwinkel-Röntgenstreuungsdatensätze überwacht, die das Vorhandensein der Wechselwirkung zwischen ComK und MecA weiter bestätigten

Structural insights into regulation of gene expression

The thesis comprises two parts investigating structural aspects of various mechanisms of translational control. The first chapter investigates a mechanism behind ribosome stalling alleviation. Poly-proline stretches often induce ribosome stalling, which needs to be alleviated to translate the mRNA. In bacteria the ribosomes are rescued by Elongation Factor P (EF-P). EF-P is activated by diverse post-translational modifications (PTMs) of a positively charged amino acid. Such PTM is the glycosylation of arginine conserved in approximately 10% of all bacterial species including severe pathogens (e.g. Pseudomonas aeruginosa). The arginine is glycosylated by a glycosyltransferase EarP which attaches rhamnose to the arginine. Impairing the glycosylation reduces the pathogenicity of the bacteria. However, EarP is an uncharacterized glycosyltranferase as it is only the first documented case of arginine N-glycosylation in prokaryotes. Hence, the mechanism of EF-P rhamnosylation by EarP was investigated using nuclear magnetic resonance spectroscopy, X-ray crystallography and various in vivo and in vitro assays. The atomic structure of EarP with its substrate dTDP-rhamnose was solved and the in vivo and in vitro assays together with subsequent studies elucidate the putative mechanism of EarP rhamnosylation thus providing basis for targeted antibiotic drug design. The second chapter investigates the translational suppression of the hunchback (hb) mRNA. The hb mRNA forms during Drosophila development a protein gradient governing the anterior-posterior body axis formation. The Hunchback protein gradient results from the suppression of hb mRNA at posterior by a complex of three proteins – Pumilio (Pum), Nanos and Brain tumor (Brat). Nanos, expressed in an opposing gradient to Hunchback, provides spatial information. Uniformly expressed Pum and Brat are RNA binding proteins that specifically recognize the hb mRNA. The structure of Brat bound to the hb mRNA, and the structure the complex of Pum and Nanos bound to the hb mRNA have been previously solved. However, it remains unclear how exactly these three proteins assemble on the hb mRNA together, and if they are structurally and functional directly linked. The complex was investigated using modelling based on small-angle X-ray and neutron scattering data combined with additional restraints from cross-linking/mass spectrometry. The data were further complemented and validated by various in vitro assays such as electrophoretic mobility shift assays and isothermal titration calorimetry. The investigation provides initial insights about the complex and paves the way for future approaches to fully elucidate the structure of the complex

EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocar

Integration and visualisation of clinical-omics datasets for medical knowledge discovery

In recent decades, the rise of various omics fields has flooded life sciences with unprecedented amounts of high-throughput data, which have transformed the way biomedical research is conducted. This trend will only intensify in the coming decades, as the cost of data acquisition will continue to decrease. Therefore, there is a pressing need to find novel ways to turn this ocean of raw data into waves of information and finally distil those into drops of translational medical knowledge. This is particularly challenging because of the incredible richness of these datasets, the humbling complexity of biological systems and the growing abundance of clinical metadata, which makes the integration of disparate data sources even more difficult. Data integration has proven to be a promising avenue for knowledge discovery in biomedical research. Multi-omics studies allow us to examine a biological problem through different lenses using more than one analytical platform. These studies not only present tremendous opportunities for the deep and systematic understanding of health and disease, but they also pose new statistical and computational challenges. The work presented in this thesis aims to alleviate this problem with a novel pipeline for omics data integration. Modern omics datasets are extremely feature rich and in multi-omics studies this complexity is compounded by a second or even third dataset. However, many of these features might be completely irrelevant to the studied biological problem or redundant in the context of others. Therefore, in this thesis, clinical metadata driven feature selection is proposed as a viable option for narrowing down the focus of analyses in biomedical research. Our visual cortex has been fine-tuned through millions of years to become an outstanding pattern recognition machine. To leverage this incredible resource of the human brain, we need to develop advanced visualisation software that enables researchers to explore these vast biological datasets through illuminating charts and interactivity. Accordingly, a substantial portion of this PhD was dedicated to implementing truly novel visualisation methods for multi-omics studies.Open Acces