Tell me how it begins, I will tell you how it ends: catching transcription at the start. To function, our cells need proteins, which are made by ribosomes, which are partly constituted of ribosomal RNAs synthesized in our cells by RNA polymerase 1 (Pol I). Pol I activity is essential during cell growth, and central in the development of many cancers. Ribosome assembly is mainly regulated at the start of ribosomal RNA synthesis by Pol I. Here, we will employ cutting edge biophysics techniques to reveal how this process is controlled one Pol I molecule at a time.

Het begrijpen van het menselijk brein is cruciaal voor hersenfunctie, neuro-ontwikkelingsstoornissen, neurale techniek en kunstmatige intelligentie. Huidige benaderingen missen echter de precisie en resolutie om te bestuderen hoe menselijke neuronen signalen verwerken en reacties produceren. Het ELEANOR-project onderzoekt de signaalverwerking van menselijke neuronen met een hoog-precieze alles-optische aanpak, waarbij een veelzijdige fotonische microchip en optische dynamische klemmen worden gebruikt voor precieze en selectieve stimulatie van individuele dendritische vertakkingen. Deze innovatieve benadering belooft huidige beperkingen in het begrijpen van de hersenfunctie van de mens aan te pakken, en de neurowetenschappen en gerelateerde vakgebieden significant vooruit te helpen.

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The 2002 outbreak of SARS and emergence of MERS in 2012 identified coronaviruses (CoV) as zoonotic threats of pandemic potential. Nevertheless, COVID19 caught us off guard. While vaccines have been produced at an incredible speed, the development of antiviral drugs is lagging. Therapeutic intervention, however, will be key to protect the unvaccinated and vaccine-non responders, to reduce mortality and the duration of hospitalization, as well as to deal with potential vaccine breakthroughs by variants or with future CoV pandemics. RNA synthesis and viral assembly are conserved pivotal steps in the CoV life cycle, and thus should offer ample opportunities for broad spectrum drugs. In CoV-infected cells, viral genomic and subgenomic RNAs are synthesized by multi-protein replication-transcription complexes (RTCs) confined to virus-induced double membrane vesicles (DMV). Viral RNAs are exported from these replication organelles into the cytoplasm through pores to become translated or, in the case of newly produced genomic RNA, to become condensed by nucleocapsid protein N and packaged into progeny virions. The development of drugs that target RNA synthesis or RNA encapsidation is hampered by lack of a mechanistic molecular understanding of these processes. They entail dynamic, long-range RNA-RNA, protein-RNA and protein-protein interactions that are difficult to capture by standard biochemical structure-function methodologies. We propose to combine our complementary expertise in single molecule biophysics, theoretical biophysics, super resolution microscopy and molecular virology to elucidate the key determinants that control RNA synthesis and RNA condensation. We will employ a diverse set of experimental and theoretical biophysics approaches to investigate how RTC composition and RTC interactions with the genomic RNA regulate RNA synthesis. Furthermore, we will dissect the mechanism of high-frequency copy-choice RNA recombination, a key driver of coronavirus evolution and a central aspect of sgRNA production. From the variety of RNA molecules synthesized by the RTC, only the genomic RNA is packaged, and we will investigate how the N protein performs selection at the molecular level. In vitro biophysical observations and theoretical biophysical models will be corroborated in vivo in the context of the infected cell by a combination of reverse genetics, mutational analysis and forced evolution. In turn, the results from in vivo analyses will guide further biophysical experimentation and modelling. Moreover, we will apply super-resolution STED microscopy and virus infection real-time imaging (VIRIM) in fixed and living infected cells to visualize RNA synthesis, translation and packaging in time and space. Specifically, we will probe the dynamic organization and composition of RTCs in relation to the viral RNAs produced and we will test our hypothesis that RNA synthesis, translation and packaging are spatiotemporally regulated and coupled, with DMV pores as central hub. Finally, we will investigate the nature of the viral RNA–N-protein macromolecular complex in infected cells and test whether liquid-liquid phase separation is a major actor of viral genome packaging. Our consortium will create a comprehensive in vitro and in vivo molecular view of CoV replication and assembly.