Human pumilio proteins use fuzzy multivalent hydrophobic interactions to recruit the CCR4-NOT deadenylase complex to
Elise B Dunshee1, Brenna A Saladin1, David J Turner2
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
Abstract:
Pumilio proteins are conserved RNA-binding proteins that control mRNAs involved in development, proliferation, and differentiation. Human PUM1 and PUM2 repress targets by recruiting the CCR4-NOT deadenylase complex through a metazoan-specific, intrinsically disordered repression domain (RD3). Here we dissect RD3 using functional assays, protein interaction assays, and crosslinking mass spectrometry. We identify multiple RD3 peptides that are sufficient for repression and binding to the CCR4-NOT complex. Crosslinking reveals numerous mutually exclusive contacts between RD3 and CCR4-NOT, consistent with a multivalent "fuzzy" binding mode in which interactions are not defined by a single sequence or structure. Sequence scrambling shows that the linear amino acid order of RD3 is dispensable, whereas its physicochemical composition, in particular aliphatic and aromatic residues, is essential for repression and CCR4-NOT binding. These findings support a model in which multivalent interactions between intrinsically disordered regions and effector complexes, governed by amino acid composition, underlie robust PUM-mediated repression and exemplify general principles by which intrinsically disordered regions recruit CCR4-NOT to regulate gene expression.
Insights
Pumilio proteins use a disordered repression domain (RD3) to control gene expression by binding the CCR4-NOT complex. Its amino acid composition, not sequence, drives this essential gene regulation mechanism.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein Structure and Function
Background:
- Pumilio proteins are crucial RNA-binding proteins regulating gene expression in development, proliferation, and differentiation.
- Human PUM1 and PUM2 proteins utilize a metazoan-specific, intrinsically disordered repression domain (RD3) to inhibit target mRNAs.
- Recruitment of the CCR4-NOT deadenylase complex by RD3 is a key mechanism for gene repression.
Purpose of the Study:
- To dissect the functional and structural properties of the Pumilio repression domain (RD3).
- To identify the specific interactions between RD3 and the CCR4-NOT complex.
- To elucidate the role of amino acid composition versus sequence in RD3-mediated repression.
Main Methods:
- Functional repression assays to evaluate RD3 activity.
- Protein-protein interaction assays to map binding sites.
- Crosslinking mass spectrometry to determine contact points between RD3 and CCR4-NOT.
- Sequence scrambling experiments to assess the importance of amino acid order.
Main Results:
- Specific RD3 peptides were identified as sufficient for both repression and CCR4-NOT binding.
- Crosslinking revealed multiple, mutually exclusive contacts, suggesting a multivalent, "fuzzy" binding mode.
- Sequence order of RD3 was found to be dispensable, while its amino acid composition (aliphatic and aromatic residues) was critical for function.
Conclusions:
- Intrinsically disordered regions (IDRs) like RD3 employ multivalent interactions governed by amino acid composition to bind effector complexes.
- This mechanism enables robust PUM-mediated gene repression.
- The findings provide general principles for how IDRs recruit CCR4-NOT to regulate gene expression.
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