Human diseases that result from defects in intron splicing could be treated with therapeutics that control specific intron splicing reactions. This is not currently possible because we lack a sufficiently detailed understanding of the mechanism of human pre-mRNA intron splicing. Pre-mRNA splicing is catalyzed by the spliceosome, which consists of 60 different proteins and five RNAs. The physical complexity of the spliceosome has hampered studies of splicing. The spliceosome is not required for the splicing of certain fungal introns that are related to human pre-mRNA introns, the self-splicing group II introns. Studies of group II intron splicing are relevant to studies of pre-mRNA splicing because group II intron splicing occurs by the same mechanism, involving two sequential transesterification reactions, used by the spliceosome. In summary, the self-splicing group II intron is a simple model system for studies of human pre-mRNA splicing. Definition of the catalytic core of the group II intron will help define the catalytic core of the spliceosome. These studies will provide basic information relevant to applied research, aimed at the discovery of novel therapeutics. A fundamental question is whether group II introns, and the spliceosome, use a single active site or two active sites to catalyze the two reactions of splicing. The investigator has developed a system that will allow him to determine the number of group II intron active sites. These studies will focus on two endonucleolytic cleavage reactions. One cleavage mimics the first reaction of splicing, the other mimics the second reaction. If the ribozyme has one active site, the same sequences should be required for both cleavage reactions. A particular intron sequence, domain 5, is required for the first reaction of splicing. Whether domain 5 is required for the second reaction is unknown. One reason for this lack of information is that mutations that block the first reaction also block the second, by default, since splicing occurs in two sequential reaction steps. To circumvent this problem the investigator will produce the two RNAs that would result if introns that lack domain 5 could complete the first step of splicing. The ability of these RNAs to complete the second reaction will be tested. These studies will determine whether domain 5 is required for the second reaction. Domain 5 binds tightly to a site located in another region of the intron. This interaction probably forms a key component of the active site that catalyzes the first reaction. Binding occurs by a novel tertiary interaction. The binding partner of domain 5 has not been located. The 3-dimensional structure of this interaction may be modeled using cross- linking, iron-EDTA and mutagenesis. These studies will characterize a novel RNA-RNA interaction that is likely to be essential for human pre- mRNA splicing. By defining the key ribozyme sequences that catalyze the two steps of group II splicing these studies will provide significant new information relevant to the design of therapeutic agents that control particular intron splicing reactions.
| Murray, H L; Mikheeva, S; Coljee, V W et al. (2001) Excision of group II introns as circles. Mol Cell 8:201-11 |
| Mikheeva, S; Murray, H L; Zhou, H et al. (2000) Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing. RNA 6:1509-15 |
| Mikheeva, S; Hakim-Zargar, M; Carlson, D et al. (1997) Use of an engineered ribozyme to produce a circular human exon. Nucleic Acids Res 25:5085-94 |
