Identification of Novel Drosophila melanogaster MicroRNAs

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Abstract
MicroRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in post-transcriptional regulation of metazoan development, homeostasis and disease. The full set of miRNAs is not known for any species and it is believed that many await discovery. The recent assembly of 15 insect genomes has provided the opportunity to identify novel miRNAs in the fruit fly, Drosophila melanogaster. We have performed a computational screen for novel microRNAs in Drosophila melanogaster by searching for phylogenetically conserved putative pre-miRNA structures. The ability of predicted novel miRNA precursors to be processed to produce miRNAs was experimentally verified in S2 cells and in several cases their endogenous expression at was validated by Northern blots. After experimental validation, the predictions were cross-checked with reference to a newly released set of small RNA sequences. Combining both datasets allowed us to identify 53 novel miRNA loci in the fruit fly genome 22 of which we had predicted computationally. This significantly expands the set of known miRNAs in Drosophila melanogaster. Most novel miRNAs contain unique seed sequences not found in other Drosophila miRNAs and are therefore expected to regulate novel sets of target genes. This data provides the basis for future genetic analysis of miRNA function and will aid the discovery of orthologous sequences in other species.