Molecular Mechanisms of Functionally Distinct Liquid-Liquid Phase Separation of Drosophila Loqs, Ago2 and dsRNA
Small RNA silencing is an essential process and has been associated with membrane-less cytoplasmic organelles (MLO), such as the processing-body (P-body). These MLOs arise though liquid-liquid phase separation (LLPS) phenomena that can also be triggered by RNA. In Drosophila melanogaster, dedicated small RNA silencing pathways exist for miRNAs and siRNAs and their biogenesis enzymes constitute separate biogenesis modules. We have observed that distinct splice variants of Loqs (a miRNA and siRNA biogenesis factor that binds double-stranded RNA), which are functinally dedicated to either the miRNA or the siRNA pathway, accumulate in distinct cytoplasmic spots. Thus partitioning to specific “phases” correlates with specific functions. We will build on our previous work with recombinant proteins but also cellular assays to reconstitute the distinct phases in vitro, address how specific partitioning is achieved and verify that the same principles act in vivo.
Klaus Förstemann has a background in the genetics and biochemistry of small RNA silencing, besides that we have a “normal fly lab”. In addition, the team works a lot with cultured fly cells. One of our strengths is the application of genome editing techniques to recapitulate genetic approaches, but also augment biochemical functionality of the proteins we study.
Michael Sattler and his team combing integrative structural biology, in particular solution NMR, and biophysical techniques, to study the structure, dynamics and molecular interactions of proteins and RNAs. Solution techniques provides unique information about conformational dynamics. To characterize molecular details linked to LLPS, they will combine solution and solid-state NMR with biophysical measurements to capture the conformational state and transitions involved at a molecular level.
Donau, C.; Späth, F.; Sosson, M.; Kriebisch, B. A. K.; Schnitter, F.; Tena-Solsona, M.; Kang, H.-S.; Salibi, E.; Sattler, M.; Mutschler, H.; Boekhoven, J. Active Coacervate Droplets as a Model for Membraneless Organelles and Protocells. Nat. Commun.2020, 11 (1), 5167. https://doi.org/10.1038/s41467-020-18815-9.
Tants, J. N.; Fesser, S.; Kern, T.; Stehle, R.; Geerlof, A.; Wunderlich, C.; Juen, M.; Hartlmüller, C.; Böttcher, R.; Kunzelmann, S.; Lange, O.; Kreutz, C.; Förstemann, K.; Sattler, M.: Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference. Nucleic Acids Res. (2017) Dec 1;45(21):12536-12550. DOI: https://doi.org/10.1093/nar/gkx886