Abstract

Group I introns are fascinating RNA molecules whose molecular architecture mediates accurate and efficient RNA splicing reactions. This project directly addresses three central issues regarding ribozymes and RNA splicing- First, how does the molecular structure of ribozymes result in catalytic activity and site-specificity? Second, what are the specific contributions of protein and RNA to catalytic ribonucleoprotein (RNP) complexes? Third, can ribozymes be developed into highly selective tools for inactivation and repair of targeted RNA molecules in mammalian cells? The development of ribozymes catalyzing-trans 5' splice site reactions has led to an explosion of information on the first step of group I splicing Insights into 3' splice site reactions have lagged because constructs for catalysis of trans 3' splice site reactions were unavailable. Progress to date includes: (i) Establishing the structural framework for 3' splice site reactions, (ii) Development and preliminary utilization of two fundamentally different ribozymes that catalyze 3' splice site reactions in trans, and (iii) Development of novel and powerful in vitro selection techniques for the analysis of 3' splice site reactions.
The Specific Aims of this project are to: (1) Optimize and characterize two novel group I ribozymes that catalyze 3' splice site reactions in trans. (2) Define the tertiary structure responsible for forward and reverse 3' splice site reactions. (3) Identify structural distinctions between self-splicing and protein-dependent group I introns. (4) Explore potential applications of group I 3' splice site ribozymes. Results will be of immediate importance to those studying splicing, ribozyme catalysis, molecular recognition, RNA structure, RNA-protein interactions, and molecular evolution. In addition, trans-acting ribozymes emerging from this project may be used for targeted RNA inactivation and repair in vivo, with important implications for basic science and gene-specific molecular therapeutics.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036981-12
Application #
2608854
Study Section
Molecular Biology Study Section (MBY)
Project Start
1989-01-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1999-11-30
Support Year
12
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405

  Publications

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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