Guthrie Lab Projects

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. We are currently addressing three fundamental questions:

1) How are the activities of the spliceosomal NTPases specifically regulated? A long-standing question has been 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 for catalytic activation of the spliceosome. We are currently using activity- and single-molecule FRET-based assays to identify the full set of molecular interactions that control this step. We are focusing on the roles of the positive activator Snu114, an EF2-like GTPase, and the proposed down-regulation of Brr2 by ubiquitination of a Prp8-interacting factor.

2)  How is mRNA splicing coupled to transcription, export and surveillance? While it is apparent that the nuclear steps in gene expression are temporally coupled, little is understood about the underlying mechanistic bases. We are employing an innovative high-throughput genetic platform to facilitate identification of quantitative genetic interactions; such Epistasis Mini-Array Profiles (E-MAPs) have proven powerful predictors of novel functional relationships. We are testing specific predictions from our ongoing analysis that suggest unexpected connections between the proteasome and the nuclear pore and 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 are determining the molecular basis of this novel signal transduction pathway using a genetically based strategy. We are also expanding our battery of stressors to identify other splicing “regulons”. More broadly, we are asking the biological impact of intron retention in ~5% of yeast genes by the quantitative analysis of each of ~250 strains engineered to contain a precise intron deletion.

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