Unravelling structural and functional communications between the Dhh1/DDX6 helicase and the Ccr4-Not deadenylase complex

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A key aspect of post-transcriptional gene expression regulation is the coordinated destruction of messenger ribonucleic acids (mRNAs). Two major mRNA decay pathways exist in the eukaryotic cell, with either a 3´-5´, or a 5´-3´ directionality. Both start with the shortening of the 3´ polyadenylate tail (poly(A) tail) by specialized enzymes (deadenylases). The major deadenylation complex in the cell is the multi-component Ccr4-Not complex. Ccr4-Not contains two deadenylases, Caf1 and Ccr4, which interact with each other and form a distinct deadenylase module. The Dhh1/DDX6 RNA helicase, which is a known mRNA decay factor, is an interactor of Ccr4-Not, which binds in close proximity to the deadenylase module. The effect of Dhh1/DDX6 on deadenylation by Ccr4-Not is, however, not investigated. In addition, structural information on the interaction of Dhh1/DDX6 with the deadenylase module is absent. Strikingly, mRNA decay and translation elongation are intimately coupled events. Molecular details, of how translation elongation dynamics are communicated to the 5´-3´ mRNA decay machinery, are, however, presently sparse. Interestingly, Dhh1/DDX6 binds directly to the ribosome and both, Dhh1/DDX6 and Caf1, have been shown to distinguish suboptimal transcripts from optimal ones, directly linking them to translational dynamics. In one part of the proposed project, I will investigate the effect of Dhh1/DDX6 on deadenylation by Ccr4-Not. To this end, I will initially map the interaction network of Dhh1/DDX6 and Ccr4-Not, in particular with the deadenylase module, with recombinantly expressed Ccr4-Not components and Dhh1/DDX6 variants. Based on these results, I will elucidate the structure of Dhh1/DDX6 in complex with the Ccr4-Not deadenylase module. Furthermore, I will delineate the functional interplay of Dhh1/DDX6 and Ccr4-Not and test the effect on deadenylation. Functional aspects of this work include in vitro deadenylation assays with recombinantly purified proteins and in vivo studies in yeast. In the second part of my proposal, I will investigate structurally the communication of the 5´-3´ mRNA decay pathway with the ribosome through direct interactions with Dhh1/DDX6. Structural studies will comprise a combination of cryo-electron microscopy, macromolecular crystallography and various mass-spectrometry methods. Results from this study will advance the fundamental understanding of post-transcriptional gene expression regulation by the major deadenylase complex, Ccr4-Not and the Dhh1/DDX6 helicase. Furthermore, this work will shed light on the intricate coupling mechanisms of translation and 5´-3´ mRNA decay.