Eukaryotic gene expression is tightly controlled to maintain proper cellular function. Eukaryotic genes are transcribed by RNA polymerase II to generate fully-processed (capped, polyadenylated, and spliced), mature messenger RNA molecules. Although the cellular machineries that carry out these reactions (RNA synthesis and RNA processing) have typically been studied as biochemically distinct reactions, they are, in fact, temporally and spatially organized to coordinately orchestrate the proper production of a fully-processed mRNA. Misregulation of any of the tightly controlled events in gene expression can have catastrophic consequences for the cell that, ultimately, lead to disease. For example, misregulation (e.g. incorrect splicing, transcriptional down-regulation, etc.) of genes encoding tumor suppressors can and does lead to cancer and a host of other human diseases. Since co-transcriptional splicing occurs in the context of a chromatin template, it is important to understand the functional links between splicing factors and chromatin-modifying enzymes. Using the model organism, Saccharomyces cerevisiae, work described here characterizes unexpected genetic interactions between the histone acetyltransferase, GCN5, within the context of an intact SAGA complex, and the U2 snRNP components MSL1 and LEA1. Furthermore, the gene encoding Gcn5, via its associated HAT activity, is required for cotranscriptional recruitment of Msl1 and Lea1 to pre-mRNAs in vivo. These studies have led to the hypothesis that Gcn5 coordinates pre-mRNA splicing with transcription through its HAT-mediated effects on transcription. To this end, the following specific aims will be undertaken: 1. Characterize the role of Gcn5 mediated histone acetylation in spliceosome assembly. Chromatin immunoprecipitation and biochemical assays will be utilized to map Gcn5 association with DBP2 and ECM33, its interactions with the U2 snRNP components, its acetylation of histones within the genes (and the functional consequence of this acetylation), and its effect on Pol II transcription. 2. Splicing sensitive microarrays will be used to identify genes whose splicing is affected by Gcn5 activity when cells are grown under normal and stress conditions. These introns will be analyzed to identify features that render their splicing Gcn5-dependent. The mechanism by which Gcn5 affects cotranscriptional splicing of these genes will be characterized by, first, mapping Msl1/Lea1 association with the genes. Then, in vivo splicing will be analyzed using the approaches described in Aim 1.
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