Investigation of the molecular mechanisms and interplay of TTP-mediated mRNA decay and translational repression using structural and biochemical tools

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This research proposal aims to understand the molecular mechanisms and interplay of mRNA decay and translational repression of ARE (AU-rich elements)-mRNA driven by the protein Tristetraprolin (TTP), using structural and biochemical tools. Post-transcriptional gene silencing is a critical step towards maintenance of a cell’s unique gene expression profile. While degradation of mRNA ensures that the level of a transcript, and eventually, its protein product is down regulated, translational repression rapidly blocks protein production without perturbing mRNA levels. Not surprisingly, translational repression has been found to precede mRNA decay in many pathways of cellular mRNA turnover. Of the known pathways of mRNA turnover, ARE-mediated decay (AMD) is especially important as many highly labile mRNAs (those of cytokines, interferons and transcription factors) contain AREs in their 3'-untranslated regions (3'-UTR) and are degraded by AMD. Consistently, AMD and the proteins involved therein are implicated in several inflammatory and autoimmune disorders and form an important platform for therapeutic intervention. A well-studied example of a protein involved in AMD is TTP. TTP specifically binds ARE-sequences and recruits the mRNA degradation machinery (the CCR4-NOT deadenylation complex and the decapping factor DCP2) to form a decay-competent mRNP. More recently, TTP was shown to mediate translational repression of its target transcript by recruiting a complex of 4EHP and GIGYF2. 4EHP, a homolog of the eukaryotic initiation factor eIF4E, interferes with translation by competing with eIF4E for binding the 5'-m7G cap and is bridged to TTP through the adaptor GIGYF2. Although the recruitment of the mRNA degradation and the translational repression machinery to the target mRNA by TTP is well understood, it is not known how or if the events of decay and translational repression are linked to each other. Furthermore, it is still unclear whether assembly of the repression machinery on the mRNA influences assembly of the decay machinery and vice-versa. To this end, we propose to use a combination of structural (NMR and X-ray crystallography), biochemical and cell-based methods to investigate the interaction of TTP with DCP2 and GIGYF2 and to analyse how these two binding events might influence each other. Since GIGYF2 was shown to impact mRNA stability, we will also explore its role in mediating decay of TTP-targets. Furthermore, we will investigate the composition of the mRNP that is assembled upon binding of TTP to its target mRNA in cells. Together these experiments will provide mechanistic insight into the dynamic assembly and remodelling of mRNPs involved in these processes and lead us to appreciate how translational repression and mRNA decay are physically and functionally coupled in cells. The proposed research will be a definite step towards understanding the interconnectivity between different steps of post-transcriptional gene regulation.