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.
Highlights
Regulated degradation of cytoplasmic mRNA is essential for the control of gene expression programmes in eukaryotic cells [1,2,3]
Reconstitution of the central module of the human Ccr4-Not complex To understand the biochemical mechanism of deadenylation by the human Ccr4-Not complex, we recently reported the reconstitution of a trimeric nuclease module composed of human BTG2-Caf1-Ccr4 [27]
The pentameric BTG2-Caf1-Ccr4-CNOT1-CNOT9 complex was reconstituted by mixing the CNOT1M-CNOT9 dimer and the trimeric BTG2-Caf1-Ccr4 nuclease module, following by a second round of Strep-affinity purification (Figure 1D)
Summary
Regulated degradation of cytoplasmic mRNA is essential for the control of gene expression programmes in eukaryotic cells [1,2,3]. Control of mRNA decay typically involves the recognition of sequence elements in the 3’ untranslated region of mRNAs by RNA-binding proteins or miRNA-mediated repression complexes, which recruit components of the RNA-degradation machinery [4,5,6]. An expanding number of RNA-binding factors have been identified that interact with the Ccr4-Not complex, which appears to be a focal point of regulated mRNA degradation. Examples include the PUF proteins [7], Nanos [8, 9] and Tristetraprolin (TTP; zinc-finger protein ZFP36) [10,11,12], which are sequence-specific RNA-binding proteins; Roquin, which binds to a stem-loop structural element [13, 14]; and GW182 (TNRC6), which is a core component of the miRNArepression complex [15, 16]
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