Investigating the role of DDX3 in regulating RNA structure and mRNA export

项目总结/摘要 DDX 3X的突变与自闭症谱系障碍、脑畸形和癫痫有关， 在不明原因的智力残疾女性病例中，这一比例高达3%。然而，目前 已知DDX 3X突变与神经发育疾病相关的分子机制。DDX3X 编码DEAD盒蛋白家族的RNA解旋酶，并与RNA的许多方面有关 然而，我们仍然缺乏对DDX 3X在这些过程中的作用的机械理解，以及如何 DDX 3X中的患者突变影响RNA代谢。 为了解决DDX 3X突变如何扰乱细胞功能的知识缺口， 并有助于神经发育疾病，我们将研究DDX 3X如何调节其机制， 在翻译起始和mRNA核输出水平上靶向转录物，并研究致病性如何 DDX 3X中的突变改变了这些过程。DDX 3X与翻译起始有关，因为它结合到 在mRNA中，5 'UTR是一个重要的翻译位点，并且是有效翻译具有结构化5' UTR的mRNA子集所必需的。但我们 目前尚不清楚DDX 3X如何调节这些mRNA的翻译。除了其 在翻译中的作用，DDX 3X已牵连在核mRNA加工，我们的初步数据显示， mRNA输出途径中的基因是DDX 3X的遗传相互作用伙伴。然而，我们仍然这样做， 不了解DDX 3X在细胞核或mRNA输出中的功能。我们假设DDX 3X 通过解旋5 'UTR中的二级结构和伴侣作用调节其靶转录物 核出口。 为了回答这个假设，在我们的第一个目标中，我们将确定DDX 3X如何影响mRNA转录 通过体内结构特异性测量RNA二级结构， 化学修饰和高通量测序。在我们的第二个目标中，我们将确定 通过使用RNA FISH染色检查DDX 3X敏感性mRNA的核输出来检测细胞核中的DDX 3X。 在成功完成拟议的实验后，我们将对 DDX 3X如何在翻译起始水平上调节RNA代谢的精确机制 作为mRNA输出。这一知识对于推进我们对RNA代谢的认识至关重要， 了解并开发DDX 3X综合征患者的治疗方法。

PROJECT SUMMARY/ABSTRACT Mutations in DDX3X are associated with autism spectrum disorder, brain malformations, and epilepsy, and account for up to 3% of cases of females with unexplained intellectual disability. However, little is currently known about the molecular mechanism linking DDX3X mutations to neurodevelopmental disease. DDX3X encodes an RNA helicase of the DEAD-box protein family and has been implicated in many aspects of RNA metabolism, yet we still lack a mechanistic understanding of DDX3X’s role in these processes, as well as how patient mutations in DDX3X affect RNA metabolism. To address this gap in knowledge of understanding of how DDX3X mutations perturb cellular function and contribute to neurodevelopmental disease, we will investigate the mechanism of how DDX3X regulates its target transcripts both at the level of translation initiation and mRNA nuclear export, and study how pathogenic mutations in DDX3X alter these processes. DDX3X is implicated in translation initiation as it binds to 5’UTRs of mRNAs, and is required for the efficient translation of a subset of mRNAs with structured 5’UTRs. However, we still do not know the mechanism of how DDX3X can regulate the translation of these mRNAs. In addition to its role in translation, DDX3X has been implicated in nuclear mRNA processing, and our preliminary data show that the genes in the mRNA export pathway are genetic interaction partners of DDX3X. However, we still do not understand what the function of DDX3X is in the nucleus or mRNA export. We hypothesize that DDX3X regulates its target transcripts through both unwinding secondary structures in their 5’UTRs and chaperoning their nuclear export. To answer this hypothesis, in our first aim we will determine how DDX3X impacts mRNA transcript structure and ribosome engagement by measuring RNA secondary structure through in vivo structure-specific chemical modification and high-throughput sequencing. In our second aim, we will determine the function of DDX3X in the nucleus by examining nuclear export of DDX3X -sensitive mRNAs using RNA FISH staining. Upon successful completion of the proposed experiments, we will have gained a greater understanding the precise mechanism of how DDX3X regulates RNA metabolism both at the level of translation initiation as well as mRNA export. This knowledge is critical for advancing our knowledge of RNA metabolism, as well as understanding and developing treatments for patients with DDX3X syndrome.