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以确定其3'端，这在添加长聚腺苷化尾部之前。没有这两个事件，mrna就不能被翻译并被破坏。因此，这个过程被称为裂解和聚腺苷化，是所有mRNA的必要事件，由称为裂解和聚腺苷化机制的蛋白质复合物控制。最近和令人振奋的证据表明，当细胞发生肿瘤时，它们表达更短的信使rna。这种缩短是一种称为选择性切割和聚腺苷化的过程的结果，通过这种过程，细胞将mRNA的3'端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节的机制尚不清楚，但可能涉及到切割和聚腺苷酸化机制或尚未发现的成员的特性的改变。在这里，我们建议开发一种能够监测聚(a)位点选择的高通量筛选试验，以识别抑制近端聚(a)位点使用的小分子。本试验将基于我的实验室开发的高度成功的转录读通报告，以监测3'端形成反应的效率。我们将与William S. Dunn化学基因组学筛选设施主任Clifford Stephan博士合作，在化学筛选中分析高度多样化的化合物库（bbb50万个独特分子）的效果。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，以区分切割和聚腺苷化的直接抑制和间接抑制，以及使用大规模平行测序方案的阳性化合物的全球影响。这些化合物将有助于研究执行这一过程的复合物的功能，也可能作为潜在的先导化合物，用于开发能够抑制癌细胞中这一过程的“智能”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.