All eukaryotes have introns in their genes. Introns interrupt coding information and must be removed after transcription through a process known as splicing. The splicing apparatus that removes these introns is highly conserved, however there are some species specific differences in the splicing apparatus that have been observed. For example, differences in splicing have been displayed between yeast and humans, and among parasitic protozoa, worms, and humans.

Since the broad functional consequences of splicing can have a dramatic impact on vitality, selectively disrupting the splicing process has medicinal potential. In order to take advantage of this potential, any disparity in splicing mechanism and regulation between organisms must be identified. Is the process of splicing biochemically distinct in specific organisms? What regulatory or structural elements can be disrupted in lower organisms without affecting mammalian splicing events?

We have constructed a GFP splicing reporter to be functional only when splicing is inhibited. We have created several control constructs and have tested the variation in GFP fluorescence in two splice mutant strains. We will use a bio-analyzer to quantitatively assess the differences in fluorescence. It may be necessary to reduce variation among individual yeast colonies by integrating the GFP reporter into the yeast genome. We are currently testing our yeast strains to visually confirm which RNA transcripts are being exported.

Using GFP as a splicing reporter in yeast, chemical screening will be carried out to explore the aforementioned possibilities. We will identify compounds that stimulate the export and translation of our unspliced yeast premessenger RNA designed so that it has an open reading frame that codes for GFP. Our hypothesis is that the nature of chemicals that disrupt splicing in yeast will provide a deeper understanding of the splicing mechanism and how it can be blocked. We will identify and characterize the chemicals that inhibit splicing. We will identify which splicing event(s) has been disrupted and by what mechanism. We will assess whether the effect will also occur in mammalian cells. The lead compounds identified by this method may provide avenues to therapeutic treatment of eukaryotic infections that are currently difficult to treat.