Expression of Large Genes: Functions and Mechanisms of Recursive Splicing in Drosophila

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

Intellectual Merit: This project will elucidate the biological roles and mechanisms of recursive splicing, a recently discovered process that is specific to genes that are interrupted by very large introns. Such introns are an important component of many vertebrate and invertebrate genomes and they must be removed accurately from the pre-mRNA transcripts during gene expression to avoid introducing mis-sense or nonsense mutations that preclude correct protein production. Extremely large introns are frequently found in genes with key roles in development and cellular regulation. Current evidence indicates that recursive splicing has a widespread role in promoting the proper expression of genes with large introns, but the underlying mechanisms remain to be elucidated. This process may facilitate efficient and accurate removal of the introns, or it may stimulate other steps in gene expression through interactions mediated by the splicing machinery. Splicing is coupled physically and functionally to transcription and other processes in mRNA biogenesis, and emerging evidence indicates that splicing itself can enhance gene expression through effects on transcript elongation, re-initiation of transcription, polyadenylation, and export of mature mRNA from the nucleus. The experimental system used in this project is the fruit fly Drosophila melanogaster. Recursive splicing has been characterized most extensively in this organism but suggestive evidence that it also occurs in higher animals has been presented. In addition, Drosophila provides powerful genetic and molecular resources for analysis of recursively spliced transcription units. The specific aims are: (1) Characterize the roles of recursive splicing in gene expression. Allele substitution techniques will be used to delete non-exonic recursive splice sites in selected but diverse genes at their native chromosomal locations. The effects on developmental phenotypes, transcription and RNA processing from the corresponding genes will be determined. (2) Characterize auxiliary elements and mechanisms for correct use of a non-exonic recursive splice site. Mutational analyses in a Drosophila cell transfection system will be used to dissect and characterize the function of sequences on the RNA that direct the use of recursive splice site RP3 in the Ultrabithorax gene. (3) Identify trans-acting factors that mediate the activity and functions of recursive splice sites. Genetic approaches will be used to identify factors required for correct recursive splicing at Ultrabithorax and frizzled and/or to mediate its role(s) in gene function. Biochemical and molecular approaches will be used to further characterize the mechanisms of identified factors. Broader impacts: This project will lead to a better understanding of pre-mRNA splicing mechanisms and strategies and their relation to other aspects of gene expression and gene structure. This is important for developing integrated models of genetic control that can have practical impact in agriculture and animal breeding, pest control, and understanding the consequences of mutation and variations in genome sequence within populations. Information generated by this project will be incorporated into an existing electronic database on recursive splicing as a publicly available resource. The project will provide research training for 2-4 graduate and 6-8 undergraduate students over a three-year period. At both levels, this will involve integrated training in experimental, computational, and comparative approaches. Three undergraduate researchers have already contributed importantly to published studies leading to this project (4 undergraduate co-authorships on 2 papers during the past two years). They have gone on to top Ph.D. programs in experimental and computational biology. Additional undergraduates will be involved, including students recruited through programs to enhance diversity. The principal investigator integrates research with education and outreach by teaching undergraduate and graduate courses in related subjects and by participating as an instructor in the Pittsburgh Supercomputing Center's Minority Access to Research Careers Summer Institute In Bioinformatics.