CAREER: Investigation of Intron Cellular Roles

University Of Toledo Health Science Campus, Toledo OH

Investigators

Abstract

Introns are ubiquitous elements of eukaryotic genomes that are especially widespread in complex organisms. The use of introns is a double-edged sword for multicellular organisms. While introns allow for complex regulation, generation of a large number of products from a limited number of genes, and facilitate emergence of new activities via exon shuffling, they also require complex processing that can lead to serious problems when it goes awry. The origins and functions of introns has been a subject of controversy for over a quarter century. In this CAREER project, the PI and his students will create or improve several databases representing gene structures, and then begin to exploit them in collaboration with three laboratories that experimentally study non-coding RNA genes, gene expression, and alternative splicing. This project will thus integrate bioinformatics, bench research, and teaching. The databases will include diverse information (location, sequence, signals, nucleotide composition, mutations, etc.) about millions of intron and exon sequences from species including unicellular eukaryotes, plants, invertebrates, non-mammalian vertebrates, and mammals. A user-friendly interface will allow a wide spectrum of queries concerning exon/intron gene structures. Computer programs that extract and process data from the databases will be developed. One set of programs, based on a comparative genomics approach, will predict, characterize, classify, and catalog non-coding RNA genes and various functional elements inside introns. Another set of programs will detect signals inside introns and exons that are important for precise splicing of primary RNA transcripts (pre-mRNA, etc.). Computer-predicted non-coding RNA genes of mammals will be experimentally verified using methods such as quantitative RT-PCR, northern blots, and large-scale hybridization with custom-generated spotted microarrays. Verification of predicted splicing motifs will be performed using two vector systems. One is the tau exon 9-11 model substrate from which a purine-rich exonic enhancer has been deleted. The second one is the caspase-2 exon 8-10 cassette from which an intronic suppressor has been removed. A novel curriculum will be created for students having two different backgrounds, computer science and biology, training them in database generation and mining, advanced programming skills, and interpretation of complex biological networks. All software developed and its documentation will be freely available via the internet and will promote remote bioinformatic education.