The Role of DEAD-box Proteins in Gene Expression

描述（由申请人提供）：真核生物中的基因表达是生物体中最复杂、高度协调的过程之一。大量研究表明，转录、信使RNA （mRNA）加工和mRNA衰变等步骤是高度耦合的，形成了一个复杂的调节蛋白质编码基因的分子回路。这一过程的核心是一类被称为DEAD-box蛋白的RNA解旋酶，它们在RNA生物学的各个方面发挥着至关重要的作用。Dbp2是酿酒酵母（Saccharomyces cerevisiae）中DEAD-box RNA解旋酶家族的一个基本未知的成员。尽管大量研究表明，Dbp2的人类同源基因hDDX5或p68具有转录调节作用，但Dbp2的转录作用尚未被描述，hDDX5在这一过程中的确切分子功能尚不清楚。我们的研究首次证明，在出芽酵母中，Dbp2是核基因表达步骤所必需的，在mRNA生物发生和染色质重塑之间发挥作用。此外，我们的工作表明，Dbp2在RNA聚合酶II合成非蛋白编码RNA的位点上起作用。在此，我们建议使用生化，分子和遗传方法的结合来详细了解Dbp2。在Aim I中，我们将利用一系列体外实验来生物化学表征Dbp2，随后分析正常细胞生长和基因表达的酶需求。在Aim II中，我们将通过分子和遗传方法的结合来确定Dbp2在基因表达中的作用。在Aim III中，我们将定义和描述使Dbp2能够“感知”新生转录本的体内分子相互作用。我们的长期工作模型是，Dbp2是基因表达回路中调节转录组的酶“开关”。因此，这些研究有可能揭示全基因组表观遗传调控的新机制，这是NIH反映当前生物医学研究面临挑战的战略倡议。重要的是，许多DEAD-box蛋白基因与人类疾病状态有关，包括癌症、神经系统疾病和艾滋病。因此，揭示单个DEAD-box蛋白的生理作用是基础生物学研究和医学界面临的重大挑战。

DESCRIPTION (provided by applicant): Gene expression in eukaryotes is one of the most complex, highly orchestrated processes in living organisms. Numerous studies have shown that steps such as transcription, messenger RNA (mRNA) processing and mRNA decay, are highly coupled, forming an intricate molecular circuit for regulation of protein-coding genes. Central to this process, are a class of RNA helicases called DEAD-box proteins that perform essential roles in all aspects of RNA biology. Dbp2 is a largely uncharacterized member of the DEAD-box RNA helicase family in the budding yeast Saccharomyces cerevisiae. Whereas numerous studies have shown that the human (h) ortholog of Dbp2, hDDX5 or p68, functions as a transcriptional regulator, no transcriptional role has been described for Dbp2 and the precise molecular function of hDDX5 in this process is not understood. Our studies now provide the first demonstration that Dbp2 is required for nuclear gene expression steps in budding yeast, functioning at the interface between mRNA biogenesis and chromatin remodeling. Furthermore, our work demonstrates that Dbp2 functions at sites of non-protein coding RNA synthesis by RNA polymerase II. Herein, we propose to obtain a detailed understanding of Dbp2 using a combination of biochemical, molecular and genetic approaches. In Aim I, we will utilize a series of in vitro assays to biochemically characterize Dbp2 and subsequently analyze the enzymatic requirements for normal cell growth and gene expression. In Aim II, we will determine how Dbp2 functions in gene expression through a combination of molecular and genetic approaches. In Aim III, we will define and characterize the in vivo molecular interactions that enable Dbp2 to 'sense' nascent transcripts. Our long term working model is that Dbp2 is an enzymatic 'toggle' in the gene expression circuit that regulates the transcriptome. Thus, these studies have the potential to reveal novel mechanisms for genome-wide epigenetic regulation, an NIH strategic initiative reflective of the current challenges facing biomedical research. Importantly, numerous DEAD-box protein genes have been linked to human disease states including cancer, neurological disorders and AIDS. Therefore, uncovering the physiological role of individual DEAD-box proteins is a major challenge to basic biological research and the medical community. PUBLIC HEALTH RELEVANCE: The ability to control the expression of specific genes is essential to cellular growth, adaptation and survival. Dbp2/hDDX5 is an RNA helicase enzyme whose over-expression promotes tumor formation and resistance to chemotherapeutics. Knowledge regarding the function of hDDX5 in gene expression is key to defining the underlying molecular basis for cancer and other human diseases.