アイテム詳細

要旨

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to degradation. The complex consists of a catalytic module comprising two deadenylases (POP2/CAF1 and CCR4) and the NOT module minimally containing the NOT1, NOT2 and NOT3 subunits. It is known that NOT1 acts as a scaffold protein for the assembly of the CCR4-NOT complex. However, the mechanism by which the NOT2 and NOT3 proteins interact with each other and dock onto the NOT1 scaffold remains unknown.NOT2 and NOT3 are related proteins that both contain a highly conserved C-terminal domain referred to as “NOT-box”. Here we show that the NOT- box is a heterodimerization domain mediating the assembly of the NOT2-NOT3 subcomplex. We have solved the crystal structures of the human NOT2 and NOT3 NOT-boxes at 2.4Å and 2.5Å resolution, respectively. The NOT-box consists of a four-stranded C-terminal open b-barrel as well as N-terminally located a-helices, which are required for heterodimerization. We also defined the domains of NOT1, NOT2 and NOT3 required for the assembly of the NOT1-NOT2-NOT3 module. Functional studies in Drosophila melanogaster cells revealed that depletion of NOT1, NOT2 or NOT3 inhibits mRNA deadenylation with a stronger effect for the NOT1 depletion, followed by NOT3. Importantly, NOT3 depletion destabilizes both NOT1 and NOT2 indicating that one important function of NOT3 is the stabilization of the NOT1 scaffold. We used mutagenesis and functional studies to identify key residues in the NOT module required for mRNA deadenylation. These studies revealed that the interaction of NOT1 with NOT2- NOT3 heterodimers is required for deadenylation in D. melanogaster cells. Similarly, NOT3 mutants that do not interact with NOT1 cannot restore deadenylation in cells depleted of endogenous NOT3. Collectively, our data shed light on the assembly of the CCR4-NOT complex and provide the basis for dissecting the role of this complex in mRNA deadenylation.