This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)
Removal of non-coding segments known as introns from precursor (pre) to messenger (m) RNAs in eukaryotic nuclei via splicing is an essential step in gene expression. The two-ste splicing reaction is carried out by a large complex of protein and RNA known as the spliceosome. Many metazoans contain two types of introns that have distinct sequences at their boundaries (GU-AG or AU-AC) and are removed by spliceosomes that contain different sets of small nuclear RNAs (snRNAs). The major class of introns is designated U2-dependent and the minor class U12-dependent according to the spliceosomal snRNA that recognizes the branchpoint. Despite a billion years of divergence, not only the fundamental chemical processes, but many of the RNA-RNA interactions required for splicing are conserved between the major and minor spliceosomes. Although significant parallels exist in the arrangement of the RNAs and in the splice site recognition events, only one snRNA is common between the two splicing systems. Recently, it was demonstrated that U4 snRNA can base pair with U6atac snRNA to splice a U12-dependent intron in vivo, raising the interesting question as to why two distinct sets of snRNAs are present in the major and minor spliceosomes. In this project, this question will be addressed by exploring finely tuned RNA-RNA interactions that are unique to the U12-dependent spliceosome. Since a number of intermolecular RNA-RNA interactions appear to be analogous and contain interchangeable functional elements between the two spliceosomes, the study will address evolutionary relationships between the major and the minor spliceosomal snRNAs. Furthermore, the mechanisms that ensure the selection of respective sets of snRNAs to form the appropriate ribonucleoprotein complexes will be defined. The research project will continue to exploit genetic suppression systems to achieve a greater understanding of the relationship between structure and function within the U12-containing spliceosome, thereby shedding light on evolutionary questions regarding the origin and retention of U12-dependent introns and the correspondings spliceosomes in the plant and animal kingdoms.
Broader impacts: The broader impacts of this research include an enhanced understanding of the origin and significance of the co-existence of two spliceosomes. Results obtained from the proposed research will be communicated to undergraduate and graduate students through lecture and laboratory courses taught by the principal investigator. The research will influence undergraduate and graduate students working on the project by introducing them to RNA molecular biology methods in an intensive, active learning environment. In terms of outreach activities, this project will host and promote research involving high school students and underrepresented minority as well as non-minority students from a community college. These students will be given the opportunity to undertake independent research studies in the laboratory, thereby enhancing their prospects for long-term careers in research.
