Comprehensive characterization of the Drosophila transcriptome

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DESCRIPTION: Our primary goal is to comprehensively identify the functional transcribed sequences, both protein coding and non-protein coding in the model organism Drosophila melanogaster. We will provide a description of the complete gene structures with transcription start sites, polyadenylation sites, and all detectable alternative transcripts. We plan to survey representative time points throughout development, a wide-variety of tissue types and well-characterized and novel cell lines. RNAs from these samples will be used for high-resolution expression profiling of the transcriptome using whole-genome tiling arrays, RNA ligase mediated Rapid Amplification of cDNA ends, RT-PCR and cDNA library construction. RT-PCR will be used to identify and isolate transcripts for rarely expressed genes of small to medium size. The cDNA libraries will be screened using a targeted approach to identify and isolate medium to large transcripts. Characterization of small RNAs (<100 bp) requires innovative strategies and we will use high-resolution (5bp) whole-genome tiling arrays and 454 sequencing. Concurrent with these studies will be a bioinformatic analysis to identify novel unannotated genes that for the first time utilizes algorithms that synthesize expression, comparative sequence and gene prediction. Further, we plan to characterize and annotate the extent of splice variation used to generate protein isoforms and identify the sequences necessary for regulated alternative splicing utilizing in vivo splicing reporter assays, RNAi and computational analysis. Finally, ncRNAs will be validated using in vivo tissue culture assays for expression and function. The scope of these studies is unprecedented and will provide the most comprehensive set of experimental evidence of transcription for any organism. As a public resource, these studies are a prerequisite for understanding normal growth and differentiation and that will aid in understanding these processes in other organisms, including humans. Drosophila models have been developed for Alzheimers, neurodegenerative diseases and cancer. In addition, genes first identified to play a role in Drosophila development are often components of conserved regulatory networks that play important roles during animal development and have been found, in humans, to contribute to the development of a variety of human cancers.