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 mRNA-formation. In the past four years, using molecular biological approaches, we have identified regions of the mRNA precursor that are required for cleavage (formation of the 3' terminus), and showed that a factor binds stably to one of these regions during the reaction. We propose a model which provides the framework for many of the experiments described here. We 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 tumor virus, SV40, as a model and will assay maturation both in vivo and in vitro. Sequences essential for each step will be identified both by mutation and by direct chemical means. To further analyze each reaction, we will: (1) identify those features of each base which are critical for processing; (2) identify those phosphates which participate in processing and determine whether they do so by RNA-protein contacts; (3) identify factors required for processing and the activation of maternal mRNAs in C. elegans; (4) identify component(s) that bind stably to the precursor during cleavage; (5) test whether processing requires recognition of the 5' end of the pre-mRNA by using a circular RNA; and (6) determine what features of the precursor are necessary for transport by injecting processing intermediates into nuclei. The experiments proposed 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 for the rational design of such drugs.
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