Dynamics and mechanisms of Argonaute proteins and the RISC complex studied with single molecule fluorescence spectroscopy
Biochemistry
Final Report Abstract
AGO proteins are versatile players in gene regulation, defense mechanisms, and RNA silencing across different organisms. Some of these AGO proteins, among them human Argonaute2 (hAGO2) and AGO from the archaeal organism Methanocaldcoccus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognized via an AGO- associated short complementary guide strand. hAGO2 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 a key component of the eukaryotic RNA interference pathway, hAGO2 takes part in the finely tuned posttranscriptional 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 in partaking in the cellular defense against foreign nucleic acids targeting DNA rather than RNA and can operate in a guide-dependent and guide-independent manner. The understanding of this process and its regulation, however, remains sparse. Combining a series of biochemical and biophysical experiments, our work with MjAgo revealed a new interaction modality termed “guide slipping” that allows this prokaryotic AGO variant to flexibly load even non-optimal DNA substrates. Biologically, this flexible loading of DNA substrates might be of mechanistic importance to allow guide-independent cleavage activity. Extensive genetic experiments showed that MjAgo is an essential gene and cannot be knocked out. Our findings can help to improve pAgos as gene editing tools and nucleic acid sensors. Employing time-resolved single-molecule FRET measurements allowed us to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observed dynamic anchoring and release of the guide’s 3’-end from the PAZ domain during the stepwise target loading process even with a fully complementary target and detected a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. We furthermore show that Dicer opens hAgo2 to allow the loading of an miRNA or siRNA duplex into AGO and that Dicer’s disordered loops form the interaction site for hAgo2. Studying the interaction of the RNA-binding protein Mex3a, which is implicated in cancer, with the miRNA-126-5p and hAgo2 revealed the overall conformational changes that are required to form this complex and elucidated the overall structural organization of this complex. The knowledge gained in our work with human Argonaute 2, Dicer and Mex3a is crucial for advancing RNAi-based applications and RNA therapeutics.
Publications
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Die erstaunliche Vielseitigkeit der Argonauten-Crew. BIOspektrum, 26(6), 603-605.
Willkomm, Sarah; Moissl, Benedikt; Michel, Henri & Grohmann, Dina
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Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2. Nature Communications, 13(1).
Willkomm, Sarah; Jakob, Leonhard; Kramm, Kevin; Graus, Veronika; Neumeier, Julia; Meister, Gunter & Grohmann, Dina
