Dynamics and mechanisms of Argonaute proteins and the RISC complex studied with single molecule fluorescence spectroscopy

• Applicant: Professorin Dr. Dina Grohmann
• Subject Area: Biophysics ; Biochemistry
• Term: from 2012 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 217845747

Argonaute (AGO) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these AGO proteins, among them human Argonaute2 (hAGO2) and AGO from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an AGO-associated short complementary guide strand. Human AGO2 and MjAGO are representatives of the eukaryotic and prokaryotic AGO clade that share a conserved four-domain architecture. However, the biological roles of eAGOs and pAGOS differ significantly. As key components of the eukaryotic RNA interference pathway, hAGO2 takes part in the finely tuned post-transcriptional regulation of up to 60% of our genes. Failure in this process can have devastating consequences ranging from diabetes and several forms of cancer to heart failure. In contrast, prokaryotic AGO proteins are implicated to partake in the cellular defence against foreign nucleic acids targeting DNA rather than RNA and are able to operate in a guide-dependent and guide-independent manner. The understanding of this process and its regulation, however, remains sparse. We have developed biochemical, structural and cutting-edge optical single-molecule methods that allow us to illuminate the structure-function-dynamics relationship of prokaryotic and eukaryotic AGO proteins. With our investigations we intend to deepen the understanding of eAGO and pAGO mechanisms and the conformational space sampled by AGO proteins throughout their activity cycle thereby shedding light on the question how these structurally conserved proteins are able to function in biologically highly diverse pathways. We will focus on the following questions:Crystal structures and functional studies of MjAGO from our laboratory indicate that MjAGO possesses a secondary nucleic acid binding channel that is occupied by a subset of substrates. We will characterise the binding channel occupancy and accompanying conformational changes. Furthermore, we aim to elucidate how complex substrates (e.g. plasmid DNA) are accommodated in prokaryotic AGOs and which regulating factors control the action of MjAGO. The activity of hAGO2 is orchestrated by various protein and nucleic acid interaction partners and modulated by post-translational modifications. How these interactors and modifications influence the structural organisation of hAGO2 is poorly understood. We aim to capture different stages of hAGO2-containing complexes to determine the structural organisation and dynamics of these complexes employing single-molecule FRET measurements. An additional major focus will be the analysis of the interaction between human AGO proteins with the pre-processor nuclease Dicer on the single-molecule level.

• DFG Programme: Research Grants