Functional genomics of evolutionary conserved long noncoding RNAs in Drosophila

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Biologists have recently discovered that a large fraction of the genomes of most organisms, previously thought to be "junk DNA", has biochemical activity and may be functional. Some of the genetic elements found in those regions of the genome do not code for proteins (the building blocks of cells) and have thus been named "non-coding RNAs". A few of these non-coding RNAs are known to play important roles in the biology of organisms, mainly by their capacity to turn other genes on or off. However, it is not clear if most of the non-coding RNAs that have been catalogued are even functional. This project seeks to address that gap in understanding by using patterns of evolutionary conservation in two fruit fly species to identify candidate non-coding RNAs for functional characterization. Because non-coding RNAs are found in many organisms, from flies to plants to humans, the results could have far-reaching impact. The work will be carried out by college students at graduate and undergraduate levels, thus providing them with valuable training experiences. In addition, the project will provide research experience and support with curriculum development for K-12 science teachers. One of the most exciting recent advances in molecular biology has been the discovery of a large number of long non-coding RNAs (lncRNAs) in the transcriptomes of eukaryotic organisms. Although about a dozen lncRNAs are known to play important roles in a broad spectrum of developmental and physiological phenomena, our understanding of lncRNA function lags significantly behind our ability to quickly catalog them using RNA sequencing and computational approaches. Given that there are thousands of lncRNAs in any given genome, one essential approach to select relevant lncRNAs for functional characterization is to focus on lncRNAs with strong patterns of evolutionary conservation. This project will focus on a group of lncRNAs that are conserved between Drosophila melanogaster and D. pseudoobscura, species that diverged from their common ancestor 25-40 million years ago. These lncRNAs show small but detectable levels of DNA sequence conservation, conserved synteny, and conserved developmental expression patterns that suggest shared function in the two species. This project will characterize the phenotypic effects of that group of evolutionary conserved lncRNAs in Drosophila. Aims will include first confirming that the conserved putative lncRNAs are independent untranslated transcripts using a combination of IsoSeq and Riboseq data, then identifying their patterns of tissue-specific expression in both species using RNAseq, and finally targeting a group of the lncRNAs for functional analysis using using transcript disruption enabled by CRISPR/Cas9 genome editing in D. melanogaster. The methods developed in this study should have broad applicability across other organisms, and the functional studies should add substantially to our understanding of what lncRNAs do.