SGER: Strategy for Systematic Identification of Targets for a Novel Splicing Pathway

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This research focuses on a novel splicing system thought to be common among eukaryotic cells but for which only a single example of a defined mRNA substrate has thus far been identified. The primary goal in this work is to develop a systematic means for identifying additional substrates for this unique splicing system. This novel splicing mechanism was first identified as a component step in a stress-response signaling pathway in the yeast Saccharomyces cerevisiae. A key step in this pathway, termed the Unfolded Protein Response (UPR), is regulated splicing of the mRNA encoding a transcriptional activator (termed Hac1p). The result is to modulate levels of the Hac1p activator which, in turn, controls expression of the cognate stress response genes. This splicing event is not mediated by the conventional mRNA splicing apparatus. Instead, two distinct gene products, Ire1p and Trl1p, act as splicing endonuclease and RNA ligase, respectively, to carry out the splicing reaction. Remarkably, the Ire1p is a transmembrane receptor that responds to changes in ligand binding by modulating its own splicing activity. Thus, splicing is truly a component step in the signaling cascade that constitutes this stress response pathway. Extensive evidence exists for conservation of UPR function among eukaryotes including the observations that yeast Hac1 mRNA can be accurately spliced in mammalian cells and recombinant human Ire1p accurately cleaves yeast Hac1 RNA. Based on this conservation of function, it is hypothesized that: (1) Regulated splicing may be a component step in UPR signaling pathways in all eukaryotes. Thus, Hac1 homologues may be found in a wide variety of eukaryotic cells. (2) This splicing system may serve a variety of gene regulatory functions other than the UPR response. Thus, regulated splicing of a variety of mRNAs may be mediated by this system. These hypotheses can be tested by the systematic identification and characterization of splicing substrates from a variety of organisms. The use of Trl1p as an affinity ligand in a selection/amplification approach will be tested as a means for conducting such a systematic analysis. The experimental goals are as follows. First, microarray analyses will be used as a comprehensive means for characterizing populations of Saccharomyces mRNAs enriched in a selection/amplification procedure. Second, application of this approach to another organism and the utility of homologous versus heterologous Trl1 gene products as affinity reagents will be assessed. Candida albicans has been chosen for this purpose based on the availability of appropriate genomic resources, its medial phylogenetic relationship to Saccharomyces, and on evidence for conservation of the splicing machinery in this organism. The results of this exploratory research will make it possible to evaluate the significance of this splicing system as a fundamental stress response mechanism among eukaryotic cells and to assess the potential for additional gene regulatory functions for this novel splicing pathway.