The long term objective of these studies is an understanding of the genetics and biochemistry of the regulation of eukaryotic messenger RNA metabolism. The current focus of these studies is the 3- untranslated region of mRNA. Two signal elements, AATAAA and downstream GT- or T- clusters, are required for efficient processing and polyadenylation. Genetic approaches are proposed to determine the optimum spacing for these two elements, and the effect on 3- RNA processing of introducing secondary structure into the 3- untranslated region of the precursor RNA. The poly(A) signal region of the mouse DHFR gene contains 4 poly(A) signals; only one contains an AATAAA, and no identifiable GT-rich and T-rich second elements are located near poly(A) sites. The DHFR poly(A) signals will be studied in order to define additional sequences which function as poly(A) signal elements. The mechanism of 3-RNA processing will be studied by both genetic and biochemical means. The patterns of polyadenylation site selection will be analyzed in transcription units containing multiple poly(A) signals; the effects on these patterns of altering the spacing between signals will be determined. The biochemistry of 3- RNA processing is being studied in nuclear extracts of Hela cells. Patterns of in vitro RNA processing of mutant substrates is being determined. Gel retardation assays and glyverol gradient analyses are being employed to monitor the assembly of substrate into a processing complex. Fractionation of extracts is planned. Both genetic and biochemical studies of mRNA stability are planned. Vectors containing inducible promoters are being developed to permit analysis of mRNA half lives. The effects on mRNA stability of changing the sequence or structure of the 3- untranslated region will be determined. Other experiments will determine how mRNA half lives are affected by changes in cellular physiology or mRNA concentration. TA-rich mRNA destabilizer sequences have been found in the 3-untranslated region of many growth factor and inflammatory response genes; experiments are planned to determine whether mRNA translation is required for these sequences to signal rapid mRNA turnover, where within an mRNA these sequences may be located and still function, and how mRNA half-lives are affected by changes in the length of the TA sequence.
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