Our broad goals going forward remain unchanged: we continue to seek molecular mechanisms for the fidelity and regulation of mRNA processing, with a particular focus on mRNA splicing in yeast. The three aims of this proposal are as follows: 1) Building on our previous demonstration that the spliceosome is a highly dynamic RNA-protein machine whose fidelity relies on RNA-dependent ATPases of the DEAD-box family, we are now employing single molecule FRET to focus on the dynamics of the ATP-dependent rearrangement most critical for the catalytic activation of the spliceosome. Brr2 is required for the unwinding of U4 from U6, allowing U6 to adopt its active conformation. By judicious placement of fluorophores in U6 RNA, which is then reconstituted into a snRNP, we will determine the directionality, step-size and processivity of unwinding and ask how these parameters are influenced by the U5 snRNP protein Prp8, which is required for Brr2-dependent unwinding. Additionally, we will test roles for post-translational modifications of Prp8, including cycles of ubiquitination and deubiquitination. 2) Our analyses using high-density genetic interaction maps have revealed evidence for an extensive interplay between splicing and transcription and, in particular, point to important roles for chromatin structure and modification We aim to parse the co-transcriptional environment by employing this large battery of double mutants in analysis of global splicing profiles by our splicing-sensitive microarrays in combination with ChIP of splicing factors and chromatin marks. We will also ask the role of polII elongation rate in splicing efficiency, exploiting an allelic series of mutations in the Trigger Lop (generated by Craig Kaplan) that alter transcription speeds over a 40-fold range. Extrapolating from the """"""""kinetic model"""""""" for changes in alternative splicing patterns in metazoa, we would predict that slowing the rate would enhance splicing by allowing more time for co-transcriptional loading of snRNPs. Interestingly, our preliminary data suggest a more nuanced and transcript-specific picture. In parallel, we will ask similar mechanistic questions in S. pombe, where the multi- intronic structure of many genes is more like that of metazoa;thus we can explicitly ask whether polII actually pauses to ensure co-transcriptional splicing, as recently suggested in budding yeast. 3) One rationale for co- transcriptional coupling is that it enables coordination of multiple steps to fine-tune gene expression in response to the immediate needs of the cell. In this aim we explore two novel instances of co-transcriptional processes that appear to rely on coupling for appropriate biological responses to extracellular cues. In one case, amino acid starvation results in a rapid and specific down-regulation of ribosomal protein gene splicing;surprisingly, this response is dependent on the identity of the promoter, not the intron. In the second, efficient expression of the PAB1 gene, which is essential for mRNA export, requires a functional connection between the locus and Brr6, an essential nuclear envelope protein. Alterations in the dynamics of transient targeting generate a bi-modal population of cells, explaining the incomplete penetrance of the brr6-1 export phenotype.
Human genes are interrupted by stretches of DNA that are read out into RNA but must be removed with a high degree of precision because failure to do so alters the resultant product, often with catastrophic consequences. It is now clear that errors in this process of RNA splicing are causative agents in a number of cancers. Our recent and future progress in determining the underlying molecular mechanisms of fidelity maintenance should provide important opportunities for potential therapeutic interventions.
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