Coordination of phase separation and translational control by the RNA helicase DDX3X

项目摘要 DDX 3X是一个重要的翻译起始因子，从酵母到人类都是保守的。解除DDX 3X管制 DDX 3X表达、DDX 3X突变或两者都与几种癌症有关，包括乳腺癌、结肠直肠癌、肺癌、口腔癌、 鳞状细胞癌和髓母细胞瘤，一种儿科脑癌。在髓母细胞瘤中，DDX 3X突变 促进细胞质信使核糖核蛋白（mRNP）颗粒（应激颗粒）的形成， 通过液-液相分离，并与全球变化的翻译。若干条证据 表明应激颗粒形成和翻译的整体变化也发生在其他癌症中， DDX 3X在连接这些过程中起着关键作用。DDX 3X如何协调翻译起始和颗粒 形成不理解。这项建议直接针对这个问题。Aim 1结合酶学 技术与荧光显微镜，以确定DDX 3X相分离和 解旋酶活性初步数据显示，与结合蛋白的牛痘病毒蛋白K7的直接相互作用， DDX 3X的N端固有无序区，破坏DDX 3X颗粒并抑制DDX 3X 酶活性将表征RNA、ATP和K7结合对DDX 3X颗粒动力学的影响 开发相分离对DDX 3X解旋酶活性影响的模型，并测试K7 破坏癌症相关的DDX 3X颗粒。目的2使用核糖体分析来表征 DDX 3X相分离和翻译控制。DDX 3X耗竭产生的翻译特征， 将比较DDX 3X颗粒形成和K7对DDX 3X颗粒的破坏，以确定DDX 3X 相分离改变了翻译，以及这些变化是否可以通过颗粒破裂来逆转。预计 这些数据将为理解mRNP颗粒形成的生物化学和酶促方面绘制新的版图。 这项工作也将为其他RNA结合蛋白的机制分析提供路线图， 有细胞质颗粒最后，由于DDX 3X催化活性的抑制促进颗粒形成， 可能驱动肿瘤发生，如髓母细胞瘤，颗粒的同时破坏， 通过K7或类似效应物抑制DDX 3X活性可能代表了治疗 与DDX 3X功能障碍有关的癌症。

Project Abstract DDX3X is an essential translation initiation factor that is conserved from yeast to human. Deregulated DDX3X expression, DDX3X mutations, or both, are linked to several cancers, including breast, colorectal, lung, oral squamous cell cancers, and medulloblastoma, a pediatric brain cancer. In medulloblastoma, DDX3X mutations promote formation of cytoplasmic messenger ribonucleoprotein (mRNP) granules (stress granules), which form by liquid-liquid phase separation and are associated with global changes in translation. Several lines of evidence suggest that stress granule formation and global changes in translation occur in other cancers as well, and that DDX3X plays a key role in linking these processes. How DDX3X coordinates translation initiation and granule formation is not understood. This proposal directly addresses this problem. Aim 1 combines enzymological techniques with fluorescence microscopy to define the relationship between DDX3X phase separation and helicase activity. Preliminary data show that direct interaction with the vaccinia virus protein K7, which binds the N-terminal intrinsically disordered region of DDX3X, disrupts DDX3X granules and suppresses DDX3X enzymatic activity. The effect of RNA, ATP, and K7 binding on DDX3X granule dynamics will be characterized to develop a model for the impact of phase separation on DDX3X helicase activity and to test the ability of K7 to disrupt cancer-associated DDX3X granules. Aim 2 uses ribosome profiling to characterize the interplay between DDX3X phase separation and translational control. Translational signatures resulting from DDX3X depletion, DDX3X granule formation, and the disruption of DDX3X granules by K7 will be compared to identify how DDX3X phase separation alters translation and whether these changes can be reversed by granule disruption. Expected data will chart new territory for understanding biochemical and enzymatic aspects of mRNP granule formation. The work will also provide a roadmap for the mechanistic analysis of other RNA binding proteins that associate with cytoplasmic granules. Finally, since inhibition of DDX3X catalytic activity promotes granule formation that may drive tumorigenesis, as is suggested for medulloblastoma, the simultaneous disruption of granules and suppression of DDX3X activity by K7 or similar effectors may represent a new strategy for the treatment of cancers linked to DDX3X malfunction.