Project Details
Projekt
Molecular mechanisms of functional liquid-liquid phase separation of Drosophila Loqs, Ago2 and dsRNA during siRNA biogenesis
Subject Area
Biochemistry
Animal Physiology and Biochemistry
Structural Biology
Cell Biology
Animal Physiology and Biochemistry
Structural Biology
Cell Biology
Term
since 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 419138605
Small RNA silencing serves to regulate genes but also as a defense against pathogens and selfish genetic elements. In insects, a specialization of small RNAs and their biogenesis factors for these tasks can be observed. Since RNA interference is a major antiviral defense pathway, functional separation and “insulation” of biogenesis factors is important to ensure a strong and rapid response to pathogens. Liquid-liquid phase separation (LLPS) appears to play an important role and miRNAs, which regulate genes, associate with P-bodies (a membrane-less organelle). It is currently unclear whether a corresponding organelle exists for siRNAs. We have investigated the phase separation based on the interaction of Drosophila Loqs, a dsRNA binding domain protein as well as the N-terminal glutamine-rich disordered domain of Drosophila Ago2 with double-stranded RNA, the precursor of siRNAs. We found that the unique Ago2 intrinsically disordered region (IDR) phase-separates together with nucleic acids. Coacervates of Ago2 with dsRNA enable co-condensation of Loqs protein, aided by its glutamine-rich region. Biophysical analysis demonstrated that these phase droplets form reversibly, that proteins remain mobile and that the RNA binding by Loqs is required to co-condensate with Ago2 droplets. NMR experiments identified that the Ago2 IDR is indeed unstructured in solution but its conformation is affected upon phase separation. The glutamine-rich region of Loqs transiently interacts with the first dsRNA binding domain, suggesting an auto-regulatory mechanism that may trigger siRNA biogenesis and phase transition concomitantly. GFP-Loqs fusion protein forms spots that retain protein mobility and exchange with the surrounding phase in Drosophila cells. The ability to bind dsRNA, in particular involving the second dsRNA binding domain of Loqs, is essential for the phase separation in cells. We propose to study the mechanistic, residue-level, details of the molecular features that trigger phase separation and Loqs / Ago2 co-condensation by combining biophysical experiments, microscopy and NMR spectroscopy in vitro, and functional studies in cells and in vivo. In particular, we will address the following questions: (i) Which molecular interactions, chemical and structural features in Loqs-PD and Ago2 mediate phase separation and client partitioning in vitro? How is a specific flux of dsRNA and/or siRNAs into the condensate achieved? (ii) How do these molecular features relate to LLPS and condensate formation in vivo? Are there additional factors that enter the phase separated droplets in vivo? (iii) How does LLPS affect silencing efficiency and small RNA profiles? Identification of key features and residues from the in vitro experiments will guide cell biology and transgenic assays with strategically designed point mutants to obtain a comprehensive description of how dsRNA-dependent phase separation influences small RNA biogenesis and silencing efficiency in vivo.
DFG Programme
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