The Role of DEAD-box Proteins in Gene Expression

Rna解旋酶是一类调节活细胞中rna结构的酶，在各个方面都有作用。 从转录到腐烂的RNA生物学。然而，绝大多数生物的精确生物学功能 约40个真核RNA解旋酶在很大程度上是未知的。在上一个资金周期中，我们提供了首批 到目前为止，一个死盒解旋酶的体内酶解旋酶靶标的鉴定。此外，我们还发现了 酿酒酵母rna聚合酶II转录终止中rna结构重塑的新作用。 这种活动受到环境信号的调节，以控制新陈代谢基因的表达，这是一个作用 我们发现它在人类细胞中是保守的，与哺乳动物的死盒解旋酶DDX5一起。引人注目的是，OBP2- 依赖的非编码转录本称为长非编码RNA(LncRNAs)，形成RNA-DNA杂交结构 或在发挥基因调控功能的Dbp2失活时产生R-环。然而，连接这些的机制(S) 多项观察结果仍不得而知。填补这一缺口至关重要，因为监管不善 许多RNA解旋酶，包括DDX5，会导致疾病状态，如神经紊乱和 癌症。剩下的主要问题是：1.依赖于Dbp2的RNA结构如何影响终止？ R环抑制和Dbp2之间的关系是什么？3.哺乳动物Dbp2，被称为DDX5， RNA重塑在终末调控中的作用？我们的中心假设是Dbp2和DDX5是 一类新的表观遗传调节器的成员，它解开新生RNA中的RNA结构以控制 转录终止和R环的形成响应于细胞的能量状态。我们建议对此进行测试 假设有三个集中的具体目标，将新建立的和创新的战略与 经过验证的实验技术。在目标1中，我们将定义RNA结构如何影响转录 终止。在目标2中，我们将确定Dbp2和R环抑制之间的机制关系。 在目标3中，我们将确定DDX5在人类细胞中的酶靶标及其与转录的联系 终止。这项研究涉及RNA生物学、基因调控和人类疾病的多个方面。

RNA helicases are a class of enzymes that modulate RNA structure in living cells, functioning in every aspect of RNA biology from transcription to decay. However, the precise biological function of the vast majority of the ~40 eukaryotic RNA helicases is largely unknown. In the previous funding cycle, we provided one of the first identifications of in vivo enzymatic helicase targets for a DEAD-box helicase to date. Moreover, we uncovered a novel role for RNA structure remodeling in transcription termination of RNA polymerase II in S. cerevisiae. This activity is regulated in response to environmental cues to control the expression of metabolic genes, a role that we found is conserved in human cells with the mammalian DEAD-box helicase DDX5. Strikingly, OBP2- dependent non-coding transcripts termed long non-coding RNAs (lncRNAs) form RNA-DNA hybrid structures or R-loops upon inactivation of Dbp2 that function in gene regulation. However, the mechanism(s) linking these multiple observations remains unknown. Filling this gap is of key importance because misregulation of numerous RNA helicases, including DDX5, results in disease states such as neurological disorders and cancer. Major remaining questions are: 1. How does Dbp2-dependent RNA structure impact termination? 2. What is the relationship between R-loop suppression and Dbp2? 3. Does mammalian Dbp2, termed DDX5, function in termination regulation through RNA remodeling? Our central hypothesis is that Dbp2 and DDX5 are members of a novel class of epigenetic regulators that unwind RNA structures in nascent RNAs to control transcription termination and R-loop formation in response to cellular energy status. We propose to test this hypothesis with three, focused Specific Aims, which integrate newly established and innovative strategies with proven experimental techniques. In Aim 1, we will define how RNA structure impacts transcriptional termination. In Aim 2, we will determine the mechanistic relationship between Dbp2 and R-loop suppression. In Aim 3, we will identify the enzymatic targets of DDX5 in human cells and connection to transcription termination. This research is relevant to multiple aspects of RNA biology, gene regulation, and human disease.