Research Summary
Eukaryotic genes are frequently interrupted by insertions of non-coding sequences, or introns. Removal of these introns by the splicing machinery is an essential step in the expression of these genes, the failure of which can lead to human disease. Given the paramount importance of excising introns precisely and accurately, it is surprising that introns contain few conserved sequences, vary widely in length and are found at dozens of locations in a single transcript. How can cells achieve exquisite precision while splicing such diverse introns? Our long-term goal is to elucidate the mechanical aspects of splicing in an effort to understand how this extraordinary machine accommodates substrate diversity while constraining errors that lead to human disease.

The splicing machinery’s RNA components, rather than protein components, play key roles in both intron recognition and removal. During splicing, these RNA components undergo extraordinary rearrangements. These RNA rearrangements may serve to constrain errors in splicing by coupling activation of the splicing machinery for catalysis with recognition of a genuine intron. An evaluation of this popular hypothesis, however, requires a mechanical understanding of the splicing machinery. RNA-stimulated ATPases of the DEAD-box family, which function in a wide range of processes including transcription, transport and translation, have been implicated in orchestrating the RNA rearrangements, but even the roles for these factors remain controversial. 

Our current goals are (1) to determine the precise function for DEAD-box proteins in turning the splicing machinery on and off and (2) to determine the folding pathway of RNA rearrangements that leads to activation and deactivation of the splicing machinery. To pursue our goals, we employ the budding yeast Saccharomyces cerevisiae, which allows for a powerful, combined approach of genetics and biochemistry. Ultimately, we expect that an understanding of how the splicing machinery is put together and taken apart will provide insights into the mechanisms that establish fidelity in splicing and prevent the splicing errors that result in human disease.