Structural and biochemical characterization of eukaryotic mRNA decapping activators

In eukaryotes, mRNA turnover starts with the truncation of 3′
poly(A) tail and proceeds with either 3′-to-5′ degradation by the
exosome complex or with decapping followed by 5′-to-3′ degradation
by Xrn1. mRNA decapping is catalyzed by the decapping enzyme
complex Dcp1-Dcp2 and is regulated by a highly conserved set of
decapping activator proteins, including Pat1, Dhh1, Edc3 and the
heptameric Lsm1-7 complex. The mechanisms regarding the interplay
of mRNA decapping activators remains elusive owing to limited
structural and biochemical understanding. My doctoral research was
focused on elucidating the structural and functional roles of mRNA
decapping activators involved in mRNA decay. Pat1 has a modular
domain architecture that allows it to interact with multiple
decapping activators simultaneously. Pat1 acts as a bridging factor
between the 3′-end and the 5′-end of the mRNA by interacting with
multiple proteins involved in decapping. The interaction of S.
cerevisiae Pat1 N-terminus with the DEAD-box protein Dhh1 was
characterized by biochemical pull-down assays and binding
affinities were determined quantitatively by isothermal titration
calorimetery. Based on these experiments, the crystal structure of
Dhh1 bound to Pat1 was determined at 2.8 Å resolution. The
structure reveals that Pat1 wraps around RecA2 domain of Dhh1 via
evolutionary conserved interactions. This conserved surface of Dhh1
is also implicated in interaction with another decapping activator,
Edc3, rationalizing why Pat1 and Edc3 binding to Dhh1 is mutually
exclusive. These interactions were supported by testing mutations
in in vitro assays with the yeast proteins and in
co-immunoprecipitation assays with the corresponding human
orthologs. Furthermore, structural analysis combined with RNA
pull-down assays and a crosslinking mass spectrometry based
approach gave definitive evidence that Dhh1 engages with Pat1, Edc3
and RNA in a mutually exclusive manner. The Lsm1-7 complex is
another important activator of mRNA decapping. It protects the mRNA
transcripts from 3′-end degradation and enhances the mRNA
decapping. I determined the crystal structure of the Lsm1-7 complex
at 2.3 Å resolution showing a hetero-heptameric complex of Lsm1-7
proteins that make a ring-like overall topology. Furthermore, an
unusual helical structure of Lsm1 C-terminal extension and
protrudes into the central channel of the heptameric ring,
explaining how it is modulates the RNA binding properties of the
complex. The Lsm1-7 complex interacts with the C-terminal domain of
Pat1. Structure determination of this octameric Lsm1-7-Pat1 complex
at 3.7 Å gave insights into the interaction of Pat1 with Lsm1-7
complex. Unexpectedly, Pat1 binds to Lsm2 and Lsm3 but not with the
cytoplasmic specific subunit Lsm1. The Pat1 C-terminus makes a
super-helical structure consisting of HEAT-like repeats of
anti-parallel helices similar to the structure of its human
ortholog. Structure based mutagenesis analysis by in vitro
pull-downs showed that these interactions are conserved. This
doctoral thesis gives structural and mechanistic insight into the
role of multi-domain protein Pat1 and how it engages at two
distinct ends of mRNA by interacting with Dhh1 at 5′-end and with
Lsm1-7 complex that at 3′-end. Combining these results present a
model of dynamic interplay of these activators and gives a better
understanding of protein-protein and protein-RNA interaction
network in the decapping machinery.