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

这项研究计划旨在了解由蛋白质Tristetraprolin（TTP）驱动的ARE（富含AU的元件）-mRNA的mRNA衰变和翻译抑制的分子机制和相互作用，使用结构和生物化学工具。转录后基因沉默是维持细胞独特基因表达谱的关键步骤。虽然mRNA的降解确保了转录物的水平以及最终其蛋白质产物被下调，但翻译抑制迅速阻断蛋白质产生而不干扰mRNA水平。毫不奇怪，在许多细胞mRNA周转的途径中，已经发现翻译抑制先于mRNA衰变。在已知的mRNA周转途径中，ARE介导的衰变（AMD）尤其重要，因为许多高度不稳定的mRNA（细胞因子、干扰素和转录因子的那些）在其3 '-非翻译区（3'-UTR）中含有战神并且被AMD降解。一致地，AMD和其中涉及的蛋白质涉及几种炎性和自身免疫性疾病，并形成治疗干预的重要平台。与AMD有关的蛋白质的一个充分研究的例子是TTP。TTP特异性结合ARE序列并募集mRNA降解机制（CCR 4-NOT去腺苷化复合物和去帽因子DCP 2）以形成有衰变能力的mRNP。最近，TTP显示通过募集4 EHP和GIGYF 2的复合物来介导其靶转录物的翻译抑制。4 EHP是真核起始因子eIF 4 E的同源物，通过与eIF 4 E竞争结合5 '-m7 G帽来干扰翻译，并通过衔接子GIGYF 2桥接至TTP。尽管通过TTP将mRNA降解和翻译抑制机制募集到靶mRNA是很好理解的，但尚不知道降解和翻译抑制事件如何或是否相互关联。此外，目前还不清楚mRNA上的抑制机制是否会影响衰变机制的组装，反之亦然。为此，我们建议使用结构（NMR和X-射线晶体学），生物化学和基于细胞的方法相结合，以调查TTP与DCP 2和GIGYF 2的相互作用，并分析这两个结合事件如何可能相互影响。由于GIGYF 2被证明影响mRNA的稳定性，我们还将探索其在介导TTP靶衰变中的作用。此外，我们将研究mRNP的组成，该mRNP在TTP与其靶mRNA结合后组装在细胞中。总之，这些实验将提供机制的洞察力的动态组装和重塑的mRNP参与这些过程，并引导我们了解如何翻译抑制和mRNA的衰变是物理和功能耦合在细胞中。这项研究将是理解转录后基因调控不同步骤之间相互联系的明确一步。

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.