The 3' ends of eukaryotic messenger RNAs are formed by cleavage of the primary transcript, followed by addition of a poly(A) tail. Polyadenylation is required for RNA stability and the efficiency by which a gene's transcript is polyadenylated directly influences the level to which its mRNA accumulates and is translated. This is exemplified by a case of alpha-thalassemia in which a single point mutation was found in the poly(A) signal of the alpha-globin gene. One goal of the proposed research is to define the structural requirements of poly(A) signals. The experimental approach involves mutagenesis of portions of poly(A) signals followed by functional assays involving transfected COS cells or HeLa cell nuclear extracts. The mouse beta maj globin and SV40 early poly(A) signals will be studied in detail, as well as a variety of hybrid and synthetic constructs. The concept of poly(A) signals as interrupted palindromes is used as a guide for many of the experiments. The second goal of the proposed research is to elucidate the rules that govern poly(A) site selection. How poly(A) sites are selected when other poly(A) or splicing signals are located on the transcript will be studied using both in vivo and in vitro approaches. The relationship between poly(A) signals and introns will be explored in detail. These studies will give insight into the regulation of differential RNA processing.
