The work in my laboratory focuses on two major steps in gene expression, mRNA splicing and mRNA export. Our long-term goal is to understand at a molecular level the mechanisms responsible for specificity and fidelity in these pathways. Our future work will address three fundamental questions: 1) How are the activities of the spliceosomal NTPases specifically regulated? A long-standing question is how the spliceosomal DEAD-box ATPases are activated at precise times in the splicing cycle. We recently identified a region of the U5 snRNP protein Prp8 that specifically stimulates the Brr2 ATPase to unwind U4 from U6 snRNA, the key event in catalytic activation of the spliceosome. Using activity- and FRET-based assays, we will now identify the full set of molecular interactions that control this step. We will focus on the roles of the positive activator Snu114, an EF2-like GTPase, and the proposed down-regulation of Brr2 by ubiquitylation of a Prp8-interacting factor. 2) How is transcription coupled to mRNA splicing and export? While it is apparent that the nuclear steps in gene expression are temporally coupled, little is understood about the underlying mechanistic bases of this coupling. We are employing an innovative high-throughput genetic platform to facilitate identification of quantitative genetic interactions; such Epistasis Mini-Array Profiles have proven powerful predictors of novel functional relationships. We will test specific predictions from our ongoing analysis that suggest unexpected connections between the proteasome and the nuclear pore, and between the spliceosome and the chromatin remodeling machinery. 3) How is splicing regulated in response to the environment? Using a global microarray-based assay, we recently demonstrated that amino acid starvation selectively inhibits the splicing of ribosomal protein gene transcripts. We will now determine the molecular basis of the novel signal transduction pathway mediating this response. We will also expand our battery of stressors to identify other splicing regulatory modules. More broadly, we will interrogate the biological impact of yeast introns by the quantitative analysis of each of ~270 strains engineered to contain a precise intron deletion. ? ? ?
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