Research in Professor Peebles' laboratory focuses on RNA processing and the mechanism of RNA splicing. Transcription usually produces RNA molecules far larger than the mature, functional RNA products that all cells need for life. Much of the RNA is removed by RNA processing. RNA processing includes RNA splicing, which excises non-coding internal parts (called introns) and assembles the useful coding sections (known as exons) into mature RNAs. RNA splicing is most common in higher eukaryotes such as animals and green plants, in which almost all genes contain many introns. Several diverse mechanisms of RNA splicing are known, and all phylogenetic kingdoms support some form of RNA splicing. RNA splicing must have appeared very early in the history of life on Earth and is as basic to genetic information metabolism as protein synthesis or DNA replication.
Dr. Peebles applies the methods of molecular biology and biochemistry to understand a particular system of RNA splicing. His group was the first to describe group II intron self-splicing, and they continue to study the structure and function of these complex RNA catalysts. Splicing in higher eukaryotes produces an excised intron as a lariat RNA that is just like group II introns. It is now thought that the ancestor of most introns and their complex splicing machinery in animals and plants was very similar to modern group II introns. Thus group II introns are the best model system for exploring the chemical mechanism of the lariat-forming pathway of RNA splicing.
The structure and function of domain 5, a small part required for the catalytic action of group II introns, has been analyzed in detail. Domain 5 was tested as a separate RNA that cooperates with a large RNA containing the rest of a group II intron. The two RNAs form a complex that performs all of the usual reactions of splicing. In this test, domain 5 is unchanged and acts repeatedly, so it can be a true catalyst. This method has been used to determine what nucleotide bases in domain 5 are needed for activity. Essentially the same sequence motif has been identified and is required for splicing in higher eukaryotic systems.
Libraries of domain 5 variants have been made in which part of the normal sequence has been randomized. The splicing reaction then selects those variants that function. This selection approach mimics evolution in the laboratory to find the best sequence for domain 5. This method can be used to analyze other parts of group II introns as well and to select for functions not in the usual repertoire of these RNA catalysts.
To learn where domain 5 interacts with the structure of the group II intron, 4-thio-uracil-modified domain 5 was made and photocrosslinked. These results show that the conserved part of domain 5 contacts another conserved sequence in the joiner between domains 2 and 3 of the intron. More crosslinking work is planned, and mutations affecting these conserved bases have been made and analyzed. These results will constrain the 3-dimensional models of active group II intron structures. In cooperation with Professor Graham Hatfull, studies of RNA processing in mycobacteria and their phages have been started. Initially, the tRNA genes found on phage L5 were changed into nonsense suppressors. This will allow collection of nonsense mutations in other phage and bacterial genes and their use as conditional alleles. A system for recombination and shuttling is being built to put site-directed mutations on the intact phage L5 genome.