Our long-term goal is to define the role for DExD/H box ATPases in pre-mRNA splicing, an essential step in the expression of eukaryotic genes that provides a versatile mechanism for regulating gene expression through splice site choice. Splicing requires eight, conserved DExD/H box ATPases. While such ATPases are thought to remodel RNA in nearly all RNA-dependent process, our understanding of the function and mechanism of this class of molecular motor proteins is poor. Toward a deeper understanding of the role of DExD/H box ATPases in splicing, we have endeavored to define their RNA targets, their mechanisms for rearranging RNA, and their roles in splice site specificity. While fidelity mechanisms have been well-defined at the stages of transcription and translation, at the stage of pre-mRNA splicing our understanding of fidelity mechanisms remains rudimentary. While the majority of spliceosomal DExD/H box ATPases have been implicated in fidelity, we lack a fundamental understanding of how splice site specificity is established by these ATPases. A major roadblock has been the lack of assays for DExD/H box-dependent rejection mechanisms. We have now successfully established assays for rejection at the catalytic stage of splicing, positioning our lab to define the fundamental basis for splicing fidelity. Further, we have recently discovered that these DExD/H box ATPases not only reject suboptimal splice sites but also enable alternative, optimal sites, thus defining these factors as alternative splicing factors that act at the unconventional stage of splicing catalysis. These findings have also yielded insight into the fundamental mechanism by which the spliceosome searches for a 3' splice site. Lastly, while a mechanistic understanding of DExD/H box ATPases in RNA-dependent processes is limited, these proteins have been commonly thought to act by destabilizing RNA-RNA or RNA-protein interactions directly. We have recently discovered evidence for an alternative mode of action in which these ATPases act indirectly by pulling on a strand of RNA to disrupt interactions at a distance. Importantly, this new mode of action would permit a DExD/H box ATPase to disrupt a duplex that would otherwise be inaccessible for direct unwinding mechanisms. To define the role and mechanism of DExD/H box ATPases at the catalytic stage of splicing, we propose to accomplish the following three aims, using a combination of single molecule, biochemical, mathematical, genetic, and genomic approaches in budding yeast. First, we aim to determine the mechanism and consequence of proofreading at the first step of splicing. Second, we aim to define the mechanism of splice site choice at the second step of splicing. Third, we aim to determine the mechanism by which DExD/H box ATPases function at the catalytic stage. This work promises to transform our understanding of the roles of DExD/H box ATPases in splicing and beyond. 1
In an intermediate stage of the expression of our genes into proteins, the workhorses of our cells, interrupting information must be accurately deleted. Errors in this process have been implicated in numerous diseases, including, blindness, neurodegenerative disease, and cancer. A major goal of our work is to determine how errors in this process are prevented to better understand the etiology of disease and how to treat disease.
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