Abstract
In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key event in regulated mRNA degradation. A major enzyme involved in deadenylation is the Ccr4-Not deadenylase complex, which can be recruited to its target mRNA by RNA-binding proteins or the miRNA repression complex. In addition to six non-catalytic components, the complex contains two enzymatic subunits with ribonuclease activity: Ccr4 and Caf1 (Pop2). In vertebrates, each deadenylase subunit is encoded by two paralogues: Caf1, which can interact with the anti-proliferative protein BTG2, is encoded by CNOT7 and CNOT8, whereas Ccr4 is encoded by the highly similar genes CNOT6 and CNOT6L. Currently, it is unclear whether the catalytic subunits work co-operatively or whether the nuclease components have unique roles in deadenylation. We therefore developed a method to express and purify a minimal human BTG2-Caf1-Ccr4 nuclease sub-complex from bacterial cells. By using chemical inhibition and well-characterized inactivating amino acid substitutions, we demonstrate that the enzyme activities of Caf1 and Ccr4 are both required for deadenylation invitro. These results indicate that Caf1 and Ccr4 cooperate in mRNA deadenylation and suggest that the enzyme activities of Caf1 and Ccr4 are regulated via allosteric interactions within the nuclease module.
Highlights
In eukaryotic cells, virtually all mature cytoplasmic mRNAs are characterized by the presence of a 3 poly(A) tail
Product analysis by denaturing PAGE indicated that these observations were not due to artefacts of the fluorescence assay (Figure 3D). These findings indicate that both Caf1 and Ccr4b are required for deadenylation by a trimeric BTG2–Caf1–Ccr4b nuclease sub-complex
The results demonstrate that (1) a complex containing Caf1 and Ccr4 is more active than its isolated components; and (2) both Caf1 and Ccr4 are required for deadenylation by a trimeric BTG2–Caf1–Ccr4 nuclease subcomplex in vitro
Summary
Virtually all mature cytoplasmic mRNAs are characterized by the presence of a 3 poly(A) tail. The shortening and removal of the poly(A) tail (deadenylation) is the initial and often rate-limiting step in regulated mRNA decay Subsequent decapping exposes both ends of the mRNA to exonucleolytic degradation involving the Xrn nuclease (5 -3 ) and/or the multi-subunit exosome complex (3 -5 decay) [3,4,5]. The nuclease subunits are tethered to the non-catalytic subunits via interactions between Caf and the central MIF4G domain of the large subunit, CNOT1 (Not1) [19,20] This subunit plays critical roles in the selective recruitment of the Ccr4–Not complex to target mRNAs as exemplified, for instance, by interactions with the RNA-binding proteins tristetraprolin (TTP) and Nanos [21,22,23,24]. In addition to selective recruitment to target mRNAs, the Ccr4–Not complex can be recruited to mRNA via interactions with the conserved N-terminal BTG domains of Tob and Tob
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