RNA splicing is a critical step in gene expression, involving excision of introns and covalent joining of exons from an RNA transcript. Specific RNA-RNA interactions are key features of splicing; they are essential for both splice site selection and catalysis of the splicing reactions. To date, studies on self-splicing group I introns have identified major structural features responsible for the catalytic activity of these ribozymes. However, two critically important and long-standing problems regarding splicing of group I introns remain unsolved. We do not know how 3' splice sites are selected; neither do we know how the catalytic core of the intron interacts with the splice sites. We have recently discovered a novel two base-pair interaction (termed P9.0) between the catalytic core of some group I introns and intron sequences at the 3' splice site, and have shown experimentally that this interaction exists and is functionally important in the exon ligation step of splicing (Burke, J.M. et al. Nature, in press). The discovery has allowed the P.I. to propose a tripartite model for 3' splice site selection (Burke, J.M. FEBS, Lett. 250, 129-133 (1989)) and puts us in a position to finally solve the 3' splice site problem. The specific objectives of this proposal are to (l) Determine the detailed structural basis for 3' splice site selection in group I introns, and (2) Use this information to develop a transacting ribozyme based on 3' splice site cleavage reactions. The project will provide insights on RNA splicing, catalysis and structure that will be of general importance to those studying splicing, biological catalysis by RNA, RNA structure, and the role of RNA structure in the mechanisms and regulation of gene expression. In addition, the ribozymes developed may have important applications as tools for in vitro manipulation of RNA, and as gene-specific agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036981-06
Application #
3291756
Study Section
Molecular Biology Study Section (MBY)
Project Start
1989-01-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Chowrira, B M; Berzal-Herranz, A; Burke, J M (1995) Novel system for analysis of group I 3' splice site reactions based on functional trans-interaction of the P1/P10 reaction helix with the ribozyme's catalytic core. Nucleic Acids Res 23:849-55
Tasiouka, K I; Burke, J M (1994) A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction. Gene 144:1-7
Berzal-Herranz, A; Chowrira, B M; Polsenberg, J F et al. (1993) 2'-Hydroxyl groups important for exon polymerization and reverse exon ligation reactions catalyzed by a group I ribozyme. Biochemistry 32:8981-6
Chowrira, B M; Berzal-Herranz, A; Burke, J M (1993) Novel RNA polymerization reaction catalyzed by a group I ribozyme. EMBO J 12:3599-605
Williamson, C L; Desai, N M; Burke, J M (1989) Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron. Nucleic Acids Res 17:675-89
Burke, J M (1989) Selection of the 3'-splice site in group I introns. FEBS Lett 250:129-33
Burke, J M (1989) Sequences and classification of group I and group II introns. Methods Enzymol 180:533-45
Burke, J M (1988) Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review. Gene 73:273-94
Been, M D; Barfod, E T; Burke, J M et al. (1987) Structures involved in Tetrahymena rRNA self-splicing and RNA enzyme activity. Cold Spring Harb Symp Quant Biol 52:147-57
Burke, J M; Belfort, M; Cech, T R et al. (1987) Structural conventions for group I introns. Nucleic Acids Res 15:7217-21

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