A HTS Assay for Inhibitors of Proximal Cleavage and Polyadenylation

描述(由申请人提供)：所有信使RNA(MRNA)必须经历成熟或加工事件来定义它们的3‘端，这是在添加一个长的多腺苷化尾巴之前。如果没有这两个事件，mRNAs无法被翻译并被破坏。因此，这一过程被称为切割和多腺化，是所有信使核糖核酸的必需事件，并由称为切割和多腺化机制的蛋白质复合体控制。最新的和具有挑衅性的证据表明，当细胞经历肿瘤发生时，它们表达较短的信使RNA。这种缩短是被称为交替切割和多腺苷基化的过程的结果，通过该过程，细胞将mRNA3‘末端的位置改变到更近的位置。这一过程如何在癌细胞中被错误调控的机制尚不清楚，但可能涉及切割和多聚腺苷酸化机制或尚未发现的成员的性质变化。在这里，我们建议开发一种高通量筛选试验，能够监测聚(A)位点的选择，以努力识别抑制近端聚(A)位点使用的小分子。这项分析将基于我实验室开发的非常成功的转录直读报告，以监测3‘端形成反应的效率。在与Te William S.Dunn化学基因组筛选设备主任Clifford Stephan博士的合作下，我们将在化学筛选中利用这种分析方法，分析高度多样化的化合物库(&gt；50,000个独特分子)的影响。最后，我们将开发反筛选来确定抑制剂的特异性，以及三个独立的二级筛选来区分直接和间接抑制切割和多腺苷化的作用，以及使用大规模平行测序方案确定阳性化合物的全球影响。这些化合物将有助于询问执行这一过程的复合体的功能，也可能作为潜在的先导化合物，开发能够在癌细胞中抑制这一过程的“智能”化疗药物。 与公共卫生相关：最近的观察表明，当细胞发生肿瘤时，它们通过交替的切割和多聚腺苷酸化的过程缩短其信使核糖核酸的长度。这项提议旨在开发一种高通量的筛选试验，以检测干扰这一过程的化合物。这项研究的成功完成将产生分子制剂，不仅将增加我们对这一过程的理解，还将作为先驱化疗药物。

DESCRIPTION (provided by applicant): All messenger RNA (mRNA) must undergo a maturation or processing event to define their 3' end, which precedes the addition of a long polyadenylated tail. Without these two events, mRNAs fail to get translated and are destroyed. This process, termed cleavage and polyadenylation, is therefore a requisite event for all mRNA and is governed by a complex of proteins called the cleavage and polyadenylation machinery. Recent and provocative evidence suggests that as cells undergo tumorigenesis they express shorter messenger RNAs. This shortening is as a result of a process known as alternative cleavage and polyadenylation whereby the cell changes the position of the 3' terminus of the mRNA to a more proximal site. The mechanism of how this is process is misregulated in cancer cells is poorly understood but likely involves alterations in the properties of the cleavage and polyadenylation machinery or yet-to-be discovered members. Here, we propose to develop a highthroughput screening assay capable of monitoring poly(A) site choice in an effort to identify small molecules that inhibit use of the proximal poly(A) site. This assay will be based upon highly successful transcriptional readthrough reporters developed in my laboratory to monitor efficiency of 3' end formation reactions. In collaboration with Dr. Clifford Stephan, Director of te William S. Dunn Chemical Genomics Screening Facility, we will utilize this assay in chemical screens analyzing the effects of a highly diverse library of compounds (>50,000 unique molecules). Finally, we will develop counterscreens to determine the specificity of inhibitors as well as three independent secondary screens to distinguish direct versus indirect inhibition of cleavage and polyadenylation as well as the global impact of positive compounds using massively parallel sequencing protocols. These compounds will be instrumental to interrogate the function of the complex that performs this process and may also behave as potential lead compounds toward the development of "smart" chemotherapeutic agents capable of inhibiting this process in cancer cells. PUBLIC HEALTH RELEVANCE: Recent observations demonstrate that as cells undergo tumorigenesis they shorten the length of their mRNA through the process of alternative cleavage and polyadenylation. This proposal aims to develop a highthroughput screening assay for compounds that interfere with this process. Successful completion of this research will generate molecular agents that will not only increase our understanding of this process but also serve as pioneer chemotherapeutic agents.