Eukaryotic RNA polymerase II transcripts are typically large, because they contain introns and extensive 3 feet flanking sequences. Removal of the 3 feet flanking sequences is an early processing step (occurs within 1 min) that involves nucleolytic cleavage at a particular site, followed by addition of poly(A) to the newly generated 3 feet end. Removal of introns by RNA splicing occurs several minutes later. RNA processing is fundamental for the expression of eukaryotic genes. The longterm goal of our research is to identify the nucleotide signals and elucidate the mechanisms by which RNA processing occurs. This information is applicable to the diagnosis of certain genetic diseases (thalassemias, for example) and potentially to their treatment.
The specific aims of the current proposal are to (1) determine the identity of the nucleotide signal that is required for accurate transcript cleavage and polyadenylation and (2) determine whether polyadenylation affects RNA splicing. A specific model for polyadenylation that involves secondary RNA structure will be tested. The experimental approach involves the modification of genes in vitro followed by assessment of their ability to produce accurately processed RNAs in transfected HeLa cells. Initial experiments will focus on mouse Beta-globin and SV40 genes. The 3 feet ends of these genes will be deleted and reconstructed in a variety of ways. The ability of the modified genes to produce, in transfected cells, transcripts that are capable of cleavage, polyadenylation and splicing will be assayed by hybridization and nucleotide sequencing techniques. Oligodeoxynucleotide synthesis will be used to construct a totally artificial signal for polyadenylation.
