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.
Highlights
MicroRNAs are small non-coding RNAs, typically 21– 23 nt long, which serve as post-transcriptional regulators of gene expression
Each primary miRNA transcript contains an extended stem-loop structure, from which a precursor pre-miRNA is released through the action of the RNAseIII enzyme Drosha and the double-stranded RNA-binding domain protein DGCR8/Pasha
The strand with the lower base-pairing energy at its 59 end is loaded onto the RNAinduced silencing complex (RISC) and guides it towards its regulatory targets [13,14]
Summary
MicroRNAs (miRNAs) are small non-coding RNAs, typically 21– 23 nt long, which serve as post-transcriptional regulators of gene expression. Based on comparison of miRNA predictions and sequence data it has been suggested that these genomes might contain many more, yet undiscovered, miRNA loci, with estimates of ,1000 miRNAs in the human and ,120 in the Drosophila melanogaster genome [6,7]. MicroRNA genes have been found as independent transcription units as well as within introns of protein coding genes. Each primary miRNA transcript (pri-miRNA) contains an extended stem-loop structure, from which a precursor pre-miRNA is released through the action of the RNAseIII enzyme Drosha and the double-stranded RNA-binding domain (dsRBD) protein DGCR8/Pasha (reviewed in [10]). The strand with the lower base-pairing energy at its 59 end is loaded onto the RNAinduced silencing complex (RISC) and guides it towards its regulatory targets [13,14]
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