ERC FUNDED PROJECTS

The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.

1 467 180 €

Start date: 2013-11-01, End date: 2018-10-31

Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.

2 177 158 €

Start date: 2019-07-01, End date: 2024-06-30

Supramolecular assemblies – formed by the self-assembly of hundreds of protein subunits – are part of bacterial nanomachines involved in key cellular processes. Important examples in pathogenic bacteria are pili and type 3 secretion systems (T3SS) that mediate adhesion to host cells and injection of virulence proteins. Structure determination at atomic resolution of such assemblies by standard techniques such as X-ray crystallography or solution NMR is severely limited: Intact T3SSs or pili cannot be crystallized and are also inherently insoluble. Cryo-electron microscopy techniques have recently made it possible to obtain low- and medium-resolution models, but atomic details have not been accessible at the resolution obtained in these studies, leading sometimes to inaccurate models. I propose to use solid-state NMR (ssNMR) to fill this knowledge-gap. I could recently show that ssNMR on in vitro preparations of Salmonella T3SS needles constitutes a powerful approach to study the structure of this virulence factor. Our integrated approach also included results from electron microscopy and modeling as well as in vivo assays (Loquet et al., Nature 2012). This is the foundation of this application. I propose to extend ssNMR methodology to tackle the structures of even larger or more complex homo-oligomeric assemblies with up to 200 residues per monomeric subunit. We will apply such techniques to address the currently unknown 3D structures of type I pili and cytoskeletal bactofilin filaments. Furthermore, I want to develop strategies to directly study assemblies in a native-like setting. As a first application, I will study the 3D structure of T3SS needles when they are complemented with intact T3SSs purified from Salmonella or Shigella. The ultimate goal of this proposal is to establish ssNMR as a generally applicable method that allows solving the currently unknown structures of bacterial supramolecular assemblies at atomic resolution.

1 456 000 €

Start date: 2014-05-01, End date: 2019-04-30

ATM is the protein kinase that is mutated in the hereditary autosomal recessive disease ataxia telangiectasia (A-T). A-T patients display immune deficiencies, cancer predisposition and radiosensitivity. The molecular role of ATM is to respond to DNA damage by phosphorylating its substrates, thereby promoting repair of damage or arresting the cell cycle. Following the induction of double-strand breaks (DSBs), the NBS1 protein is required for activation of ATM. But ATM can also be activated in the absence of DNA damage. Treatment of cultured cells with hypotonic stress leads to the activation of ATM, presumably due to changes in chromatin structure. We have recently described a second ATM cofactor, ATMIN (ATM INteractor). ATMIN is dispensable for DSBs-induced ATM signalling, but ATM activation following hypotonic stress is mediated by ATMIN. While the biological role of ATM activation by DSBs and NBS1 is well established, the significance, if any, of ATM activation by ATMIN and changes in chromatin was up to now completely enigmatic. ATM is required for class switch recombination (CSR) and the suppression of translocations in B cells. In order to determine whether ATMIN is required for any of the physiological functions of ATM, we generated a conditional knock-out mouse model for ATMIN. ATM signaling was dramatically reduced following osmotic stress in ATMIN-mutant B cells. ATMIN deficiency led to impaired CSR, and consequently ATMIN-mutant mice developed B cell lymphomas. Thus ablation of ATMIN resulted in a severe defect in ATM function. Our data strongly argue for the existence of a second NBS1-independent mode of ATM activation that is physiologically relevant. While a large amount of scientific effort has gone into characterising ATM signaling triggered by DSBs, essentially nothing is known about NBS1-independent ATM signaling. The experiments outlined in this proposal have the aim to identify and understand the molecular pathway of ATMIN-dependent ATM signaling.

1 499 881 €

Start date: 2012-02-01, End date: 2018-01-31