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.
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.
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