Although synthesis of RNA and its processing, including splicing, have historically been studied as biochemically distinct reactions, these processes are, in fact, spatio-temporally coupled such that RNA splicing takes place in the context of chromatin. This has lead to the prediction that specific chromatin marks may influence splicing. Our preliminary analyses of both yeast and mammalian transcriptomes implicate histone H3K36 methylation as a key player in splicing regulation in both systems. Here we leverage the power of yeast genetics and molecular biology to gain fundamental mechanisms into the coordination of chromatin modification and splicing, which we will further analyze in mammalian immune cells. The question of how chromatin influences splicing is particularly prescient in light of the observation that splicing primarily occurs while pre-mRNAs are associated with chromatin, suggesting that some factor(s) help retain pre-mRNAs to chromatin and only release the mRNA once splicing is completed. We propose the conceptually innovative idea that spliceosome disassembly is coupled to the state of the chromatin. Based upon preliminary data and strong collaborations with UCLA colleagues with expertise in bioinformatics, immunology, and mammalian alternative splicing, we have developed innovative tools to address the hypothesis that chromatin, particularly histone H3K36 methylation (H3K36me), and Prp43?s interaction with it affect co- transcriptional splicing Aim 1. Determine the relationship between Set2 dependent H3K36me and RNA splicing in yeast Aim 2. Determine how the yeast protein Prp43 affects co-transcriptional splicing outcomes Preliminary yeast studies lead to a number of hypotheses that can be tested in HSC-derived macrophages, in which the methyltransferase SETD2 or the factor that tethers mammalian Prp43, to the spliceosome has been knocked out using CRISPR-Cas9. With this system we will:
Aim 3. Determine how histone H3 methylation and/or mammalian Prp43 (DHX15) interaction with chromatin affects splicing outcomes and macrophage biology
Proper expression of information in genes is absolutely critical for correct cellular function; defects in any of the reactions involved in gene expression can have catastrophic consequences for the cell. Genes contain long stretches of interrupting information (introns) that must be removed by the cell in order for that information to be properly expressed, and while the cell has evolved elegant mechanisms for intron removal (a process called RNA splicing), mutations that cause defects in these mechanisms are a leading cause of a variety of human diseases?from neurodegeneration and blindness to all known cancers. The focus of our work is to understand the molecular details underlying RNA splicing as this is a crucial step toward understanding the etiology of human diseases and, ultimately, for developing tools to treat them.
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