Our long-term objective is to elucidate networks of eukaryotic small RNAs and their regulatory targets. We are motivated in this endeavor by the growing body of evidence that demonstrates fundamental cellular activities for diverse classes of RNAs in the 20-30 nucleotide range. One class that has received particular attention are the microRNAs, an abundant family of -22 nucleotide inhibitory RNAs that derive from hairpin precursor transcripts. It is estimated that there at least one thousand human microRNAs, which collectively regulate at least 30% of human genes. Additional classes of processed small RNAs include endogenous small interfering RNAs (siRNAs), repeat associated siRNAs, and Piwi-associated RNAs. We propose herein a focused set of experimental and computational experiments to characterize the small RNA component of the Drosophila melanogaster genome. (1) We will perform high-throughput pyrosequencing of a broad variety of libraries to obtain comprehensive coverage of expressed Drosophila small RNAs. (2) We will exploit the recent availability of a dozen sequenced fly genomes to make highly specific computational predictions of microRNA genes and microRNA targets in silico. (3) We will use microarray profiling and other molecular techniques to validate the endogenous expression of microRNAs and other novel small RNA genes. We will also perform in vivo phenotypic assays to demonstrate their biological activity. (4) We will elucidate the structures of primary microRNA transcripts by genetically stabilizing these transient species, followed by genome tiling microarray analysis. This work will culminate in a thorough annotation of the different classes of processed small RNAs and their associated precursor transcripts in Drosophila. Going beyond Drosophila, it is well documented that misregulation of small RNA pathways has tremendous adverse consequences for the development and physiology of all eukaryotic species. This comprehensive effort to uncover small RNA genes in flies will not only inform the annotation of small RNA genes in the human genome, but also lay the foundation for future studies of their normal and pathological roles. Insights gained from this genetically tractable model organism will be relevant for understanding the functional contributions of small RNAs to human disease.
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| Gerstein, Mark B; Rozowsky, Joel; Yan, Koon-Kiu et al. (2014) Comparative analysis of the transcriptome across distant species. Nature 512:445-8 |
| Brown, James B; Boley, Nathan; Eisman, Robert et al. (2014) Diversity and dynamics of the Drosophila transcriptome. Nature 512:393-9 |
| Mohammed, Jaaved; Flynt, Alex S; Siepel, Adam et al. (2013) The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution. RNA 19:1295-308 |
| Smibert, Peter; Miura, Pedro; Westholm, Jakub O et al. (2012) Global patterns of tissue-specific alternative polyadenylation in Drosophila. Cell Rep 1:277-89 |
| Ladewig, Erik; Okamura, Katsutomo; Flynt, Alex S et al. (2012) Discovery of hundreds of mirtrons in mouse and human small RNA data. Genome Res 22:1634-45 |
| Westholm, Jakub O; Ladewig, Erik; Okamura, Katsutomo et al. (2012) Common and distinct patterns of terminal modifications to mirtrons and canonical microRNAs. RNA 18:177-92 |
| Bejarano, Fernando; Bortolamiol-Becet, Diane; Dai, Qi et al. (2012) A genome-wide transgenic resource for conditional expression of Drosophila microRNAs. Development 139:2821-31 |
| Axtell, Michael J; Westholm, Jakub O; Lai, Eric C (2011) Vive la différence: biogenesis and evolution of microRNAs in plants and animals. Genome Biol 12:221 |
| Chung, Wei-Jen; Agius, Phaedra; Westholm, Jakub O et al. (2011) Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans. Genome Res 21:286-300 |
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