Eukaryotic mRNAs are not replicas of the genes that encode them. Instead, they are produced by a series of post-transcriptional modifications of a primary transcript. In principle, each maturation step provides a means of regulating gene expression. In the first period of this grant, my lab identified regions of the mRNA precursor that are required for cleavage (formaton of the 3' terminus), and showed that a factor binds stably to one of these regions during the cleavage reaction. From these studies, we propose a model that provides the framework for many of the experiments described here. I plan an intensive analysis of three mRNA processing steps: cleavage, polyadenylation, and transport from the nucleus to the cytoplasm. We will use the mRNA of a tumor virus, SV40, as a model, and will assay maturation both in vivo, using frog oocytes and cultured monkey cells, and in vitro, using a nuclear extract. Sequences in the precursor essential for each of these maturation steps will be identified both by mutation and by direct chemical methods. Our model predicts that any mutations affecting polyadenylation will affect cleavage identically. To further analyze each reaction, we will (1) identify component(s) (enzymes, snRNAs) that bind to the precursor during cleavage, (2) examine the products of the cleavage reaction in biochemical detail, (3) test whether processing requires recognition of the 5' end of the mRNA precursor by using a circular RNA, and (4) determine what features of the precursor are required for transport by injecting mRNA processing intermediates directly into nuclei. The experiments proposed will elucidate fundamental mechanisms of gene expression, and therefore will have important practical applications. Because various organisms use similar but distinct mechanisms to process their mRNAs, the opportunity exists to create a new generation of antibiotics directed against a previously unexploited target - mRNA processing. Our work will provide fundamental knowledge needed for the rational design of these drugs.
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