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bioRxiv · 10.64898/2026.07.16.738963

GW182 silencing domain engages the tristetraprolin-binding pocket of CNOT1 to recruit CNOT1 into multiprotein condensates

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

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. SignificanceGW182 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.

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Bialobrzewski, M. K., Cieplak-Rotowska, M. K., Michas, A., Staszalek, Z., Sonenberg, N., Dadlez, M., Niedzwiecka, A.. 2026-07-17. GW182 silencing domain engages the tristetraprolin-binding pocket of CNOT1 to recruit CNOT1 into multiprotein condensates. https://doi.org/10.64898/2026.07.16.738963

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