The RING ®nger protein CNOT4 is a component of the CCR4±NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin±protein ligase (E3). We show that the unique C 4 C 4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s). Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction. CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2±E3 interface abolish this activity. Based on these results, we present a model of how E3 ligase function within the CCR4±NOT complex relates to transcriptional regulation.

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