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The central region of CNOT1 and CNOT9 stimulate deadenylation by the Ccr4 Not nuclease module

Pavanello, Lorenzo; Hall, Benjamin; Airhihen, Blessing; Winkler, Gerlof Sebastiaan

Authors

Lorenzo Pavanello

Benjamin Hall

Blessing Airhihen



Abstract

Regulated degradation of cytoplasmic mRNA is important for the accurate execution of gene expression programmes in eukaryotic cells. A key step in this process is the shortening and removal of the mRNA poly(A) tail, which can be achieved by the recruitment of the multi-subunit Ccr4-Not nuclease complex via sequence-specific RNA binding proteins or the microRNA machinery. The Ccr4-Not complex contains several modules that are attached to its large subunit CNOT1. Modules include the nuclease module, which associates with the MIF4G domain of CNOT1 and contains the catalytic subunits Caf1 and Ccr4, as well as the module containing the non-catalytic CNOT9 subunit, which binds to the DUF3819 domain of CNOT1. To understand the contributions of the individual modules to the activity of the complex, we have started to reconstitute sub-complexes of the human Ccr4-Not complex containing one or several functional modules. Here, we report the reconstitution of a pentameric complex including a BTG2-Caf1-Ccr4 nuclease module, CNOT9 and the central region of CNOT1 encompassing the MIF4G and DUF3819 domains. By comparing the biochemical activities of the pentameric complex and the nuclease module, we conclude that the CNOT1-CNOT9 components stimulate deadenylation by the nuclease module. In addition, we show that a pentameric complex containing the melanoma-associated CNOT9 P131L variant is able to support deadenylation similar to a complex containing the wild type CNOT9 protein.

Journal Article Type Article
Publication Date Nov 9, 2018
Journal Biochemical Journal
Print ISSN 0264-6021
Electronic ISSN 1470-8728
Publisher Portland Press
Peer Reviewed Peer Reviewed
Volume 475
Issue 21
Pages 3437–3450
APA6 Citation Pavanello, L., Hall, B., Airhihen, B., & Winkler, G. S. (2018). The central region of CNOT1 and CNOT9 stimulate deadenylation by the Ccr4 Not nuclease module. Biochemical Journal, 475(21), 3437–3450. doi:10.1042/bcj20180456
DOI https://doi.org/10.1042/bcj20180456
Keywords Ribonuclease; Deadenylase; Caf1/CNOT7; Mg2+ dependent enzyme; RNA; RQCD-1; Protein expression
Publisher URL http://www.biochemj.org/content/475/21/3437

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