Bialobrzewski, M. K.; Cieplak-Rotowska, M. K.; Michas, A.; Staszalek, Z.; Sonenberg, N.; Dadlez, M.; Niedzwiecka, A.

GW182 recruits the CCR4-NOT deadenylase complex through its CIM1 region, yet the CNOT1 surface mediating this interaction has remained unknown. Using hydrogen-deuterium exchange mass spectrometry, we map CIM1 binding to a hydrophobic groove within CNOT1 residues 800-999. Unexpectedly, this groove coincides with the tristetraprolin (TTP)-binding pocket. CIM1 and the C-terminal TTP peptide engage this surface through a shared RL(P/{zeta})X{Omega} sequence pattern despite lacking overall homology, suggesting a convergently evolved short linear motif (SLiM). We further show that the GW182 silencing domain (GW182 SD) and TTP compete for CNOT1 binding using orthogonal in vitro assays, including a reconstituted liquid-liquid phase separation system in which GW182 SD acts as a scaffold and recruits CNOT1 as a client through specific binding. These findings define a shared CNOT1 recognition site and reveal SLiM-mediated competition as a molecular principle governing CCR4-NOT engagement, with consequences for GW182-driven silencing condensate assembly in vitro.