The goal of this project is to identify small molecule inhibitors of spliceosomal splicing of pre-mRNAs in mammalian cells. Splicing is an essential and highly regulated step in mammalian gene expression. There are currently no known small molecule inhibitors of splicing. To identify such compounds, we will adapt a previously published dual gene reporter assay for pre-mRNA splicing to a high-throughput configuration. We will develop dual reporter plasmids for both the major 112-dependent splicing pathway and the minor U12-dependent splicing pathway found in mammalian cells. In order to validate the comstructs for high- throughput screening, we will test them on a small molecule library available in our institution. Secondary screens of compounds which appear to inhbit splicing will include tests on cells containing constructs which do not contain introns and RT-PCR assays to evaluate the effect of the compounds on intron retention in vivo. Compounds that are inhibitory only to intron-containing reporters will be further tested for splicing inhibition in in vitro splicing assays specific to each of the two spliceosomal splicing mechanisms. The primary and secondary assays will identify small molecule compounds that inhibit spliceosomal splicing in an in vivo context. Potential uses of such compounds include 1) the biochemical investigation of the splliceosomal splicing pathways in mammalian cells, 2) investigations of the in vivo consequences of down regulation of spliceosomal splicing in intact cells, tissues and organisms, 3) investigations of the possible different roles in gene expression and gene function of the two classes of spliceosomal introns in higher eukaryotes. Public health relevance: The regulated expression of genes is central to human growth, development, normal and pathological functioning and the response of the body to changes in the internal and external environment. An important point of gene regulation is the removal of introns from the primary transcripts of genes by RNA splicing. This proposal is to discover drug-like chemical compounds that can directly influence the process of splicing. Such compounds will improve our ability to study and understand the process of splicing and may also have useful biological properties for the study or treatment of disease.
