The complex programs of gene expression involved in development, differentiation, and control of cell proliferation require sophisticated genetic regulatory mechanisms. This involves controls both at the level of mRNA formation and also at the level of mRNA turnover. Although much is known about regulatory signals located upstream from coding sequences, the 3 feet untranslated region of messenger RNA has received much less attention. The studies proposed here are directed at understanding processing and polyadenylation signals, which determine which sequences within a transcription unit will appear in the 3 feet untranslated region, and the role played by 3 feet untranslated regions in controlling mRNA concentrations. Specifically, these studies should provide answers to the following questions: 1) What is the complete signal for processing and polyadenylation of mRNAs? Is the sequence 5 feet-AATAAA-3 feet alone sufficient to signal polyadenylation? What other oligonucleotides can substitute for AATAAA? What rules govern signal choice when multiple polyadenylation signals are located in a transcription unit? Do different polyadenylation signals function with different efficiencies? 2) What sequences (or structures) within an mRNA affect its stability? What range of half-lives is seen among a series of mRNAs differing primarily in their 3 feet untranslated regions? Are there sequences within mRNAs which encode the ability of the half-life of that mRNA to change in response to specific physiological or pharmacological signals? To address these questions, a large number of gene constructions will be prepared, differing from one another primarily in sequences downstream from the coding region. Some constructs will contain polyadenylation signals from different sources. Others will contain random fragments of procaryotic or eucaryotic DNA or synthetic oligonucleotides, downstream from coding sequences. Patterns of transcription, concentrations of messenger RNA, and mRNA half-lives will be determined during the period of transient expression in cells transfected by these constructs.
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