Mechanisms of mRNA substrate-selection by the Ccr4-Not deadenylase complex

Abstract

The level to which genes are expressed depends on the rate at which the mRNA is generated, and the rate at which it is utilised and destroyed. Almost all eukaryotic mRNAs contain a stretch of adenosine nucleotides known as the poly(A) tail. The removal of the polyA tail from an mRNA, a process called deadenylation, is an important mechanism of gene expression regulation. It is the first step in the decay of the transcript, and is also linked to repression of translation. Deadenylation is predominantly catalysed by a conserved multi-protein complex called Ccr4-Not. While the poly(A) tail is a feature of almost all mRNAs, cells control the rate at which each undergoes decay by the precise targeting of Ccr4-Not in both a gene-dependent and a context-dependent fashion. Substrate-selective deadenylation is therefore a central biochemical process to the control of gene expression. It plays a pivotal role in most cellular processes including differentiation, cell cycle control and adaptation to environmental change. The inflammatory response and embryogenesis are two systems in which deadenylation has been well studied. The subject of this dissertation is the biochemical mechanisms by which mRNAs are selected for deadenylation by Ccr4-Not. Despite its importance, intact Ccr4-Not has not previously been obtained in sufficient quantity and purity for rigorous biochemical and structural analysis. Here I present the purification of recombinant Ccr4-Not. An experimental system was devised to quantify the rate and pattern of the deadenylation reaction that it catalyses in vitro. Two models of Ccr4-Not regulation were characterised in detail: the recruitment of Ccr4-Not by RNA-binding adaptor proteins, and the effect of the protein Pab1, which binds to the poly(A) tail. These have yielded insight into the features of the proteins and RNA sequences that are critical to deadenylation. In addition, a structural study of the Ccr4-Not complex was performed using electron cryomicroscopy and single-particle analysis.