Structural and biochemical characterization of the c-terminal module of the yeast Ccr4-Not complex

mRNA turnover begins with deadenylation wherein the poly(A) tail at the 3’ end of the mRNA is enzymatically removed. Deadenylation also happens to be the rate-limiting step of the decay pathway. In vivo, deadenylation is carried out by two macromolecular complexes, namely the Pan2-Pan3 complex and the Ccr4-Not complex. The Ccr4-Not complex is a multi-protein complex that is evolutionarily conserved in all eukaryotes and is considered as the major deadenylase complex in the cell. In S. cerevisiae, the Ccr4-Not complex is composed of nine subunits and is built around the scaffolding protein Not1. Structurally, the Ccr4-Not complex assembles into four separate modules with distinct domains of Not1 acting as a scaffold for individual modules. The four modules include the N-terminal module, the deadenylase module, the Caf40 module and the C-terminal module. With the exception of the C-terminal module, the architecture and biochemical role of all other modules of the yeast Ccr4-Not complex has been characterized. My doctoral thesis is focused on the elucidation of the architecture of the C- terminal module of the yeast Ccr4-Not complex. The C-terminal module can be divided in to two sub-modules, the Not module and the ubiquitylation module. The Not module is composed of the C- terminal domain of the Not1, the Not2 and the Not5 proteins in S. cerevisiae. Using limited proteolysis, the minimal core of the Not module was identified to be formed of the C-terminal domain Not1 (Not1C), full-length Not2 and the C- terminal domain of Not5 (Not5C). The minimal core of the Not module was reconstituted, crystallized and the structure was determined at 2.8 Å resolution. The structure reveals that Not1C adopts a HEAT repeat architecture with 10 HEAT repeats. The C-terminal Not-box domains of Not2 and Not5 adopt a Sm-like fold and heterodimerize via a non-canonical dimerization interface. This heterodimerization of Not2 and Not5 brings their N-terminal extended regions in proximity to each other. The N-terminal extended regions of Not2 and Not5 interact with Not1C synergistically. Loss of Not1 interacting region of either Not2 or Not5 leads to complete disassembly of the Not module in vitro and in vivo. Analysis of the electrostatic surface potential of the Not1C-Not2-Not5C crystal structure shows the presence of a positive patch on the surface. Using biochemical assays and cross-linking mass-spectrometry approaches, the RNA binding properties of the Not module were explored. The Not module binds specifically to poly(U) RNA with a major site on the Not-box domain of Not5. The ubiquitylation module consists of the C-terminal domain of Not1 and Not4. Not4 harbors a N-terminal RING domain with E3 ubiquitin ligase activity and a C-terminal low-complexity region essential for its association with the Ccr4-Not complex. I characterized distinct regions of yeast Not4 structurally and biochemically, with their respective interaction partners. First, the crystal structure of the RING domain of Not4 in complex with the Ubc4 was determined. Ubc4 is the cognate E2 enzyme of the Not4 E3 ligase. The structure of the E2-E3 complex provided insights into the specificity of Ubc4 towards Not4. Second, the minimal Not1 interacting region of Not4 was mapped and the minimal core of the Not1-Not4 complex was crystallized. Analysis of the crystal structure of Not1C in complex with the minimal interacting region of Not4 (Not4C) identified a yeast specific short linear motif in Not4c that is essential for Not1 binding. Thus, the structure provides insights into the putative differences between yeast Not4 and its homologues from higher eukaryotes that highlight the differences in the complex formation property. In brief, my doctoral thesis provides insights into the architecture of the Not module and the ubiquitylation module of the Ccr4-Not complex. Together, these results present a structural model for the C-terminal arm of the yeast Ccr4-Not complex and also provide insights into how the C-terminal module contributes to mRNA and protein degradation