Seminarium Zakładu Biofizyki

GW182 is an intrinsically disordered protein that plays a central role in post-transcriptional gene silencing. Through its N-terminal Ago-binding domain (ABD), GW182 interacts with Argonaute (Ago) in the microRNA-induced silencing complex (miRISC), while its C-terminal silencing domain (SD) recruits the CCR4–NOT deadenylase complex to targeted transcripts. CCR4–NOT is also engaged by tristetraprolin (TTP), another intrinsically disordered RNA-binding protein that directs decay of cytokine mRNAs through AU-rich elements. Although GW182 and TTP employ distinct mechanisms, their shared reliance on CCR4–NOT raises the question of whether the pathways converge or compete.Here, we combine fluorescence correlation spectroscopy studies and liquid-liquid phase separation (LLPS) experiments to investigate how GW182 SD interacts with a fragment of the central subunit of the CCR4–NOT deadenylase complex, CNOT1(800-999). We show that GW182 SD undergoes liquid–liquid phase separation with a lower critical solution temperature (LCST)-type behaviour driven by tryptophan-mediated π–π interactions. The GW182 SD forms multiprotein condensates with CNOT1, suggesting a scaffold–client interaction. We show that single-point mutations in CNOT1 distrupt this assembly. By integrating FRET with FRAP, we discriminate between proteins that are engaged in specific complexes and those retained within condensates based on residual interactions, providing a strategy for selectively probing molecular interactions in crowded environments.Importantly, the presence of a TTP peptide fragment responsible for the CNOT1 binding as a third component inhibits the formation of GW182 SD–CNOT1 condensates, indicating direct competition for the same binding region on CNOT1. Together, these findings reveal that GW182 SD not only drives LLPS and contributes to P-body assembly but also exploits the same binding site of CNOT1 as TTP, uncovering a molecular cross-talk between distinct post-transcriptional silencing pathways.