The long-term goal of the proposed research is to obtain a detailed understanding of the molecular events involved in the formation of the 3' ends of polyadenylated mRNAs in mammalian cells. These include the actual pre-mRNA cleavage-polyadenylation reaction that generates the polyadenylated mRNA 3' end, degradation of the downstream product of the cleavage reaction, and subsequent termination of transcription by RNA polymerase II, which occurs at poorly defined sites hundreds, or even thousands, of base pairs 3' to the site of polyadenylation. During the course of these studies, a model proposing a linkage between all three of these reactions will be tested.
The specific aims of the proposal are: 1) Purification and characterization of factors involved in the poly(A) site cleavage reaction. This reaction requires at least three separable factors, which will be purified, and their interactions with each other, with the pre mRNA, and with poly(A) polymerase will be studied. cDNAs for and antibodies against one or more of these factors will be produced to facilitate studies on the structure and function of these molecules. 2) Purification and characterization of the poly (A) polymerases involved in pre mRNA polyadenylation. A non-specific poly (A) polymerase appears to function together with a specificity factor, also involved in the cleavage reaction, to catalyze specific (ie, AAUAAA-dependent) polyadenylation. This enzyme will be purified and its interaction with other factors studied. Again, cDNAs and antibodies will be produced to facilitate this analysis. 3) Investigation of the role of downstream sequences (ie, sequences located 3' to AAUAAA) in cleavage and polyadenylation. Factors that interact with these sequences will be identified and their role in the polyadenylation reaction studied. 4) Purification and characterization of 5' -> 3' exoribonuclease. Downstream products of the pre mRNA cleavage- polyadenylation reaction are rapidly degraded in vitro and in vivo. A 5' - > 3' exonuclease that catalyzes these reactions in vitro has been identified. This enzyme will be purified and characterized and its role in mRNA metabolism studied. 5) Analysis of transcription termination by RNA polymerase II. It has now been shown in several instances that efficient transcription termination requires a functional mRNA polyadenylation signal. The cis-acting sequences required for termination will be further characterized. The mechanism of the reaction will be studied by developing a coupled DNA-dependent transcription- polyadenylation-termination in vitro system.
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