The central role of splicing in the eukaryotic gene expression pathway and the critical function of snRNAs in the workings of the spliceosome are well known. But unlike RNA polymerase and the ribosome, the spliceosome is known only by low-resolution views whose elements are poorly connected to function. Many open questions remain such as how do RNA helicase proteins promote RNA rearrangements during assembly of the spliceosome, and how do splicing regulatory proteins control these assembly events? To unravel the mysteries of spliceosome assembly, its regulation and its integration at the systems level, we will employ the experimentally versatile budding yeast splicing machinery as a model with three specific aims. In the first aim we will determine how the RNA helicase family member Prp5 and the RNA-binding proteins Cus2 and Mud2 promote rearrangement of pre-mRNA and U2 during prespliceosome assembly. These three proteins have RNA binding domains, but which exact RNA sequences they bind are not known. We will capture and sequence snRNA and pre-mRNA regions that bind them as splicing complex formation proceeds.
This aim will answer important questions about how the pre-mRNA is brought into the spliceosome during assembly. In the second aim we will learn how Mer1 promotes splicing through its intronic enhancer. We will apply biochemical methods including reconstitution of in vitro splicing reactions, as well as structural approaches in collaboration with Daniel Pomeranz Krummel (Brandeis) and a single-molecule method in collaboration with Melissa J. Moore (HHMI/U. Mass) and Jeff Gelles (Brandeis).
This aim will define the molecular events of splicing activation and provide a mechanistic basis for understanding more complex splicing regulation in mammalian cells. In the third aim we will explore the mechanism of trans-competition control as a new paradigm for regulation of splicing and mRNA metabolism at the systems level. Our work on the global regulation of splicing during meiosis revealed the phenomenon of pre-mRNA competition for the splicing machinery, and bears critically on the mechanism of splicing regulation at a cellular level. We will test the hypothesis that stable binding of the U2 snRNP is the competitive step.
This aim will begin to define the parameters and mechanisms of pre-mRNA competition in splicing regulation. The combined strengths of genetics, biochemistry, and genomics available in yeast and the fundamental conservation of the splicing machinery indicate that our efforts will translate directly to new understanding of the mechanisms of global posttranscriptional gene regulation in all eukaryotic cells.
Splicing is a fundamental step in the process of gene expression. Aberrant splicing leads to a multitude of human diseases ranging from myotonic dystrophy to nervous system dysfunctions to cancers. The baker's yeast Saccharomyces cerevisiae is a simple eukaryote that has proven to be a powerful model for studying fundamental aspects of splicing and its regulation. Discoveries made using yeast are readily testable in more complex mammalian systems.
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