Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation

Abstract

The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.

dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

“…Although we cannot exclude the requirement of NOT4 and CAF40 on the basis of RNAi depletion experiments, our results confirm previous observations that CAF1, NOT1, NOT2, and NOT3 are required for the general deadenylation mechanism of mRNA in Drosophila and that NOT4 and CAF40 do not seem necessary for this process. Moreover, NOT4 was not detected as a stable component of the CCR4-CAF1-NOT complex (31).…”

“…In Drosophila, CAF1 has been identified as the main enzyme involved in the general deadenylation of mRNAs (Ref. 31 and references therein). Because we previously described that CAF1 was necessary for the second deadenylation phase and for the decay of ARE mRNAs in Drosophila (23), we determined whether the intrinsic catalytic activity of CAF1 was required for the deadenylation of CecA1 mRNA.…”

miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

“…6c). We find it interesting that in yeast and in fly, the CCR4-NOT complex is known to interact with the translational repressor Dhh1/Me31b 30,31 , whose orthologs in other organisms are known to be required for miRNA-mediated repression [32][33][34] , suggesting a possible mechanism by which the CCR4-NOT complex could repress translation.…”

Developmental functions of piRNAs and transposable elements

“…55 Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, we found that nos mRNA degradation depends on its deadenylation by the CCR4-NOT deadenylation complex. 56,57 This results from the recruitment of the CCR4-NOT complex onto nos mRNA by Smg. 58 Deadenylation by the CCR4 deadenylase is also a major mechanism of RNA silencing by the miRNA pathway.…”