Abstract
<jats:title>Abstract</jats:title> <jats:p> 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/ζ)XΩ 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 <jats:italic>in vitro</jats:italic> 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 <jats:italic>in vitro</jats:italic> . </jats:p> <jats:sec> <jats:title>Significance</jats:title> <jats:p>GW182 silencing domain binds the tristetraprolin pocket of CNOT1 and recruits it into multiprotein condensates, providing a biophysical framework for coordinated miRNA- and ARE-mediated gene expression regulation.</jats:p> </jats:sec>