The broad aim of this proposal is to understand the role of the RNA polymerase II CTD. More specifically, Dr. Corden plans to test the hypothesis that the CTD is involved in the post-transcriptional processing of pre-mRNA. This hypothesis is based on their recent discovery of a set of rat proteins that interact with the CTD both in a yeast two-hybrid assay and in vitro. These CTD-binding proteins are all related to a class of serine/arginine-rich SR proteins that have been shown to be involved in both constitutive and regulated splicing. The interaction of the CTD with a set of SR proteins suggests that the CTD may act as a physical link between the transcription and processing machinery. This unexpected finding implies that the CTD could play a role in gene regulation by modulating the post-transcriptional fate of pre-mRNA. In preliminary experiments, the Corden lab has isolated full length cDNAs for four novel rat SR proteins that interact with the CTD in a two- hybrid assay. The CTD-binding domains of these proteins fall into two different and unrelated classes. They propose to perform functional studies on a subset of these CTD-binding proteins, initially focusing on S1 and X3 which are the prototypes of the two distinct CTD-binding protein families. They propose to search for the presence of these CTD- binding proteins in complexes with RNA polymerase II and test the role of this potential interaction through mutational analysis of the CTD and CTD-binding proteins. In addition, they will continue the search for additional CTD-binding proteins in both mammals and the yeast, Saccharomyces cerevisiae, where they can apply genetic approaches to the study of the role of CTD-interacting proteins. Specifically, Dr. Corden proposes work in six related areas. In part A, he proposes to determine whether the mammalian CTD-binding proteins they have identified bind the CTD in mammalian cells by isolating transcription complexes from cells and determining the composition of the complexes with antibodies to RNA polymerase II and the CTD-binding proteins. In part B, he proposes to examine the effect of CTD mutations on mRNA processing in vivo. In mouse cells, Dr. Corden plans to use his transient transfection approach in which the endogenous polymerase is inhibited by amanitin. This allows examination of the effects of CTD truncations that are normally lethal. He also proposes to examine effects of both lethal and non-lethal CTD mutations in yeast and will make use of an rpb1-1 ts mutation in the largest subunit of the endogenous RNA polymerase to examine the function of the introduced CTD mutation. In part C, he proposes to test CTD-binding proteins for splicing function. This experiment will look for the ability of the CTD-binding proteins to complement the splicing activities in an S100 extract as has been seen for other SR proteins. In part D, he proposes to characterize the stability and specificity of the interaction between CTD-binding proteins and the CTD in in vitro binding assays. In part E, he proposes to identify and characterize additional mammalian CTD- binding proteins. In part F, he proposes to identify and characterize yeast CTD-interacting proteins.
