The Role of DEAD-box Proteins in Gene Expression

RNA解旋酶是调节活细胞中RNA结构的一类酶，在各个方面发挥作用 从转录到衰变的RNA生物学过程。然而，绝大多数人的确切生物学功能 约40种真核RNA解旋酶在很大程度上是未知的。在上一个融资周期，我们提供了第一批 迄今为止，DEAD盒解旋酶的体内酶解旋酶靶标的鉴定。此外，我们发现， RNA结构重塑在S.啤酒。 这种活动是受调节的环境线索，以控制代谢基因的表达，一个角色， 我们发现在人类细胞中与哺乳动物的DEAD盒解旋酶DDX 5是保守的。值得注意的是，OBP 2- 依赖性非编码转录物称为长链非编码RNA（lncRNA），形成RNA-DNA杂交结构 或在基因调控中起作用的Dbp 2失活后的R环。然而，将这些联系起来的机制 多个观察结果仍然未知。填补这一空白至关重要，因为 许多RNA解旋酶，包括DDX 5，导致疾病状态，如神经障碍， 癌剩下的主要问题是：1。Dbp 2依赖的RNA结构如何影响终止？2. R环抑制和Dbp 2之间的关系是什么？3.哺乳动物Dbp 2，称为DDX 5， 通过RNA重塑终止调节功能？我们的中心假设是Dbp 2和DDX 5是 一类新的表观遗传调节因子的成员，它们解开新生RNA中的RNA结构， 转录终止和R-环形成。我们打算测试一下 假设有三个重点明确的目标，将新制定的创新战略与 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Dbp 2和R环抑制之间的机制关系。 在目标3中，我们将确定DDX 5在人类细胞中的酶靶点以及与转录的关系。 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.