The long term goal of the project is to understand the physical and functional connections between the mRNA elongation and processing. Although the existence of these connections is widely accepted, their nature, particularly for RNA splicing, remains to be elucidated. In the first part of this proposal we study a protein that may hold a key to a subset of these connections, the transcription elongation factor CA150. The second part of the application broadens its scope by proposing to establish in vitro and in vivo systems for the study of the functional connections between elongation and splicing. The studies proposed are divided into the following specific aims: 1. To identify and characterize the cellular targets of CA150 action. RNAi-mediated CA150 knockdowns coupled to en masse transcript analysis suggest that up to 3% of genes are regulated by CA150. We propose to identify genes that are repressed or activated by CA150 and the cis-acting elements responsive to CA150. 2. To characterize the mechanism of CA150 action. We will use both in vitro and in vivo approaches in mammalian and drosophila cells to unravel the site of CA150 action on cellular targets and the transcriptional events affected. 3. To identify and characterize functional connections between transcription and splicing of nascent transcripts. We and others have identified associations between CA 150 and the splicing machinery. Using an in vitro system we developed to couple transcription to splicing we will address the function of CA 150 and other potential connectors. We also propose to unravel the mechanism by which transcription elongation impacts on regulated alternative splicing. The importance of gene expression in health and disease is clear and we hope that completing the studies proposed here will lead to a fuller understanding of these two critical steps in gene expression and their intersection. ? ?
