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.