The role of the RNA-associated proteins NOT4 and Bam in mRNA decay

Abstract

The multi-subunit CCR4-NOT complex is an important regulator of gene expression in eukaryotes. It affects most steps of the messenger RNA (mRNA) lifecycle, but as the major deadenylase in cells, its most studied function is the enzymatic removal of the 3’-poly(A) tail of mRNAs. In the past years, several studies were carried out to shed light on how the CCR4-NOT complex is directed to specific mRNA targets, and to identify proteins that regulate this process. The main aim of my doctoral work was to study metazoan-specific aspects in the assembly of the CCR4-NOT complex and to understand molecular details of its recruitment to mRNA by mRNA-associated proteins. The NOT4 E3 ubiquitin ligase is highly conserved in eukaryotes and has been shown to associate with the CCR4-NOT complex in budding yeast. Biochemical and structural studies also demonstrated that the C-terminal region of yeast NOT4 interacts with the C-terminal part of NOT1. In contrast, different studies indicated that NOT4 is not a stable component of the CCR4-NOT complex in human (Hs) and Drosophila melanogaster (Dm) cells. During my doctoral studies, I examined the interactions of human NOT4 with components of the CCR4-NOT complex. My studies indicate that NOT4 directly interacts with the NOT1 and CAF40 subunits of the deadenylase complex in vitro. The interaction of Hs NOT4 with CAF40 is mediated by a conserved CAF40-binding motif (CBM). In addition, Hs NOT4 elicits 5’-to-3’ decay of bound mRNAs through its interaction with CCR4-NOT. Importantly, depletion of CAF40 abolishes the interaction between NOT4 and the CCR4-NOT complex and impairs the ability of NOT4 to elicit decay of bound mRNAs. In a collaborative effort among different members of the laboratory, we have also identified a CBM in the D. melanogaster protein Bag-of-marbles (Bam). As in the case of NOT4, Bam CBM allows the recruitment of the CCR4-NOT complex via CAF40. Thus, in the absence of CAF40, Bam does not interact with the CCR4-NOT complex. The findings described in this thesis support a model of targeted deadenylation where RNA-associated proteins recruit the CCR4-NOT complex to specific mRNAs, resulting in the deadenylation, translational repression and degradation of a wide range of transcripts. NOT4 and Bam bind to the same surface of CAF40 as a previously characterized RNA-associated protein, Roquin, which highlights CAF40 as an important platform for the regulated recruitment of the CCR4-NOT complex.