The Role of DEAD-box Proteins in Gene Expression

描述(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(信使RNA)加工和信使RNA衰变等步骤是高度耦合的，形成了调控蛋白质编码基因的复杂分子电路。这一过程的中心是一类被称为DEAD-BOX蛋白的RNA解旋酶，它们在RNA生物学的各个方面都扮演着重要的角色。DBP2是萌芽酵母中DEAD-box RNA解旋酶家族中一个基本未知的成员。虽然大量的研究表明，人类(H)Dbp2的直系同源基因hDDX5或p68具有转录调节功能，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也不清楚。我们的研究现在第一次证明了Dbp2是萌芽酵母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，我们的工作证明了Dbp2在RNA聚合酶II的非蛋白质编码RNA合成的位点上发挥作用。在这里，我们建议结合生化、分子和遗传学的方法来详细了解Dbp2。在目标I中，我们将利用一系列的体外实验来确定Dbp2的生化特征，并随后分析正常细胞生长和基因表达所需的酶。在AIM II中，我们将通过结合分子和遗传方法来确定Dbp2如何在基因表达中发挥作用。在目标III中，我们将定义和表征体内使Dbp2能够‘感测’新生转录本的分子相互作用。我们的长期工作模式是，Dbp2是调节转录组的基因表达回路中的一个酶“触发”。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，这是美国国立卫生研究院的一项战略倡议，反映了当前生物医学研究面临的挑战。重要的是，许多死盒蛋白基因与包括癌症、神经疾病和艾滋病在内的人类疾病状态有关。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。 公共卫生相关性：控制特定基因表达的能力对细胞生长、适应和生存至关重要。DBP2/hDDX5是一种RNA解旋酶，其过表达促进肿瘤的形成和对化疗药物的耐药性。了解hDDX5在基因表达中的功能是确定癌症和其他人类疾病潜在分子基础的关键。

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.