Molecular Mechanisms that Control mRNA Decapping in Biological Condensates

项目总结 细胞将生化反应组织成生物凝结物。P小体是保守的细胞质 凝析油富含对mRNA储存或降解重要的因素，但这些相反的结果是如何 可能在凝析油中实现的效果尚不清楚。MRNA降解的一个关键步骤是去除5‘-7- 脱壳酶复合体(Dcp1/Dcp2)形成的甲基鸟苷帽，先于并允许消化 由保守的外切核糖核酸酶形成的信使核糖核酸体。我们重组的生物冷凝物含有 裂解酵母Dcp1/Dcp2和解帽蛋白增强子3(Edc3)是P-杆菌的主要核心蛋白。 身体。使用新颖的、基于活性的荧光探针，我们有了两项重大发现。第一，相反 对于流行的凝析油由于局部浓缩效应而促进酶反应的模型，我们发现 与稀溶液相比，这种相分离抑制了Dcp1/Dcp2的活性100倍。第二， EDC3可以挽救这些凝析油的脱壳活性。我们的数据表明蛋白质之间的相互作用 Platform Dcp1是短线性蛋白质相互作用基序的集成者，它将相分离与 通过促进Dcp1/Dcp2的构象变化到自动抑制的构象来使解帽失活。 在目标1中，我们将确定Dcp1/Dcp2的自抑制构象的结构并检验假设 Dcp2中的短线状基序直接与Dcp1相互作用，促进Dcp1的瞬时不活跃构象 解封情结。在目标2中，我们将研究凝析油如何为脱壳提供额外的一层 抑制，测试促进相分离的假设相互作用进一步稳定不活跃的 凝析油中Dcp1/Dcp2的构象在目标3中，我们将确定激活的机制 在凝析油中解封，测试Edc3反对短线性不作为的抑制作用的假设 Dcp2中的基序，并促进构象变化，打开Dcp1/Dcp2中的RNA结合通道 促进凝析油内高效脱碳。对体外去顶抑制很重要的损伤将是 在分裂酵母中测试它们在EDC3介导的mRNA衰退中的作用。拟议的研究将准备好 提供一个生物缩合如何与酶的构象控制相结合的范例 在信使核糖核酸衰变水平上影响基因表达保真度的活性。

PROJECT SUMMARY Cells organize biochemical reactions into biological condensates. P-bodies are conserved cytoplasmic condensates enriched in factors important for mRNA storage or degradation, but how these opposing outcomes may be achieved in condensates is unclear. A critical step in mRNA degradation is the removal of the 5'-7- methylguanosine cap by the decapping enzyme complex (Dcp1/Dcp2) that precedes and permits digestion of the mRNA body by conserved exoribonucleases. We have reconstituted biological condensates containing fission yeast Dcp1/Dcp2 and an enhancer of decapping protein 3 (Edc3), which are major core proteins of P- bodies. Using novel, activity-based fluorescence probes we have made two significant discoveries. First, contrary to the popular model that condensates enhance enzymatic reactions due to local concentration effects, we find that phase separation represses the activity of Dcp1/Dcp2 100-fold compared to dilute solution. Second, the decapping activity of these condensates can be rescued by Edc3. Our data suggest the protein interaction platform Dcp1 is an integrator of short-linear protein interaction motifs that couples phase separation to inactivation of decapping by promoting a conformational change in Dcp1/Dcp2 to an autoinhibited conformation. In Aim 1, we will determine the structure of the autoinhibited conformation of Dcp1/Dcp2 and test the hypothesis that short-linear motifs in Dcp2 directly interact with Dcp1 to promote a transient inactive conformation of the decapping complex. In Aim 2, we will study how condensates provide an additional layer for decapping repression, testing the hypothesis interactions that promote phase separation further stabilize the inactive conformation of Dcp1/Dcp2 in condensates. In Aim 3, we will determine the mechanism for activation of decapping in condensates, testing the hypothesis that Edc3 opposes the inhibitory action of short-linear inaction motifs in Dcp2 and promotes a conformational change that opens the RNA binding channel in Dcp1/Dcp2 to promote efficient decapping within condensates. Lesions important for repression of decapping in vitro will be tested for their function in EDC3-mediated mRNA decay in fission yeast. The proposed studies are poised to provide a paradigmatic example of how biological condensation is coupled to conformational control of enzyme activity that affects the fidelity of gene expression at the level of mRNA decay.