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Sharif, Humayun (2014): Structural and biochemical characterization of eukaryotic mRNA decapping activators. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



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.