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

项目摘要 DDX3X是一种重要的翻译起始因子，从酵母到人类都是保守的。解除管制的DDX3X 表达、DDX3X突变或两者都与几种癌症有关，包括乳腺癌、结直肠癌、肺癌、口腔癌 鳞状细胞癌和髓母细胞瘤，这是一种儿童脑癌。在髓母细胞瘤中，DDX3X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，并与翻译的全球变化有关。有几条证据 提示应激颗粒的形成和翻译的整体变化也发生在其他癌症中，并且 DDX3X在连接这些过程中起着关键作用。DDX3X如何协调翻译启动和粒度 队形是不被理解的。这项建议直接解决了这个问题。目标1结合了酶学 用荧光显微镜技术确定DDX3X相分离和 解旋酶活性。初步数据显示，与痘苗病毒蛋白K7直接相互作用，它结合了 DDX3X N-末端固有无序区，打乱DDX3X颗粒，抑制DDX3X 酶活性。RNA、ATP和K7结合对DDX3X颗粒动力学的影响将被表征 建立相分离对DDX3X解旋酶活性影响的模型，并测试K7对DDX3X解旋酶活性的影响 破坏与癌症相关的DDX3X颗粒。目标2使用核糖体图谱来表征相互作用 DDX3X相分离和平移控制。DDX3X耗尽导致的翻译签名， 将比较DDX3X颗粒的形成和K7对DDX3X颗粒的破坏，以确定DDX3X如何 相分离改变了翻译，以及这些改变是否可以通过颗粒破坏而逆转。预期 数据将为理解mRNP颗粒形成的生化和酶方面绘制新的领域。 这项工作还将为分析与之相关的其他RNA结合蛋白提供路线图 有细胞质颗粒。最后，由于DDX3X催化活性的抑制促进了颗粒的形成 可能会导致肿瘤的发生，就像髓母细胞瘤所建议的那样，颗粒和 K7或类似效应物对DDX3X活性的抑制可能代表着一种新的治疗策略 与DDX3X故障有关的癌症。

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.