The Role of DEAD-box Proteins in Gene Expression

摘要 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。在前一个融资周期中，我们利用了一个强大的 多学科方法提供RNA解旋酶Dbp2的生物学和生化特征 酿酒酵母。我们特别关注这种酶是因为人类Dbp2的同源基因DDX5是 功能神秘的致癌基因。这些研究揭示了rna解旋酶在细胞中的新作用。 并阐明了对lncRNA和R-loop生物学的及时见解。然而，关键问题仍然存在。 关于Dbp2/hDDX5的精确功能，以及由此引申出来的~40死盒的所有功能 解旋酶。这些问题是：1.Dbp2针对的RNA结构是什么？2.Dbp2是如何意义的 细胞的营养状态？3.单个RNA解旋酶如何调节特定基因的表达 网络？我们的中心假设是Dbp2调节特定RNA PolII的二级结构 促进终止和抑制R环形成的转录本，以响应营养的可获得性。这个 本应用的目的是确定Dbp2是如何将营养状况与代谢基因调节相结合的 这是我们先前洞察力的直接延伸。我们建议用三个有重点的具体目标来检验这一假设， 它将新建立和创新的战略与经过验证的实验技术相结合。在目标1中，我们 将定义精确的RNA底物，用于Dbp2的酶重排。在目标2中，我们将确定如何 Dbp2的亚细胞定位受营养状态的调节。在目标3中，我们将确定 Dbp2和lncRNAs调控代谢基因的机制。这项研究涉及多个方面 核糖核酸生物学、基因调控和癌症。

ABSTRACT 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 utilized a powerful multidisciplinary approach to provide biological and biochemical characterization of the RNA helicase Dbp2 in S. cerevisiae. We specifically focused on this enzyme because the human ortholog of Dbp2, DDX5, is functionally enigmatic oncogene. These studies revealed a novel role for an RNA helicase in cellular metabolism and elucidated timely insights into lncRNA and R-loop biology. However, key questions remain regarding the precise function of Dbp2/hDDX5 and, by extension, the functions of all of the ~40 DEAD-box helicases. These questions are: 1. What are the RNA structures that Dbp2 targets? 2. How does Dbp2 sense the nutritional status of the cell? 3. How does a single RNA helicase regulate a specific gene expression network? Our central hypothesis is that Dbp2 modulates the secondary structure of specific RNA Pol II transcripts to promote termination and repression of R-loop formation in response to nutritional availability. The objective of this application is to determine how Dbp2 couples nutritional status to metabolic gene regulation as a direct extension of our prior insights. 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 the precise RNA substrates for enzymatic rearrangement by Dbp2. In Aim 2, we will determine how subcellular localization of Dbp2 is regulated in response to nutritional status. In Aim 3, we will determine the mechanism for metabolic gene regulation by Dbp2 and lncRNAs. This research is relevant to multiple aspects of RNA biology, gene regulation, and cancer.