Abstract
The Drosophila olfactory system is highly stereotyped in form and function; olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) always appear in the same antennal location and the axons of OSNs expressing the same OR converge on the same antennal lobe glomeruli. Although some transcription factors have been implicated in a combinatorial code specifying OR expression and OSN identity, it is clear other players remain unidentified. In hopes of mitigating the challenges of genome-wide screening, we examined the feasibility of a two-tiered approach comprising a primary “pooling” screen for miRNAs whose tissue-specific over-expression causes a phenotype of interest followed by a focused secondary screen using gene-specific RNAi. Since miRNAs down-regulate their targets, miRNA over-expression phenotypes should be attributable to target loss-of-function. It is the sequence-dependence of miRNA-target pairing that suggests candidates for the secondary screen. Since miRNAs are short, however, miRNA misexpression will likely uncover non-biological miRNA-target relationships. Rather than focusing on miRNA function itself where these non-biological relationships could be misleading, we propose using miRNAs as tools to focus a more traditional RNAi-based screen. Here we describe such a screen that uncovers a role for Atf3 in the expression of the odorant receptor Or47b.
Highlights
The transgenic RNAi fly stock libraries (e.g., the Vienna Drosophila RNAi library[1] and the Transgenic RNAi Project (TRiP)) have been a tremendous boon to the Drosophila community because they permit tissue-specific knockdown of almost all genes in the genome
After identifying miRNAs whose over-expression induces a phenotype of interest, bioinformatic target prediction provides a list of candidate genes for a follow-up RNAi screen
Since miRNAs inhibit the translation of or induce degradation of their target mRNAs, phenotypes observed upon miRNA over-expression should be replicable via RNAi knockdown of the responsible target(s)
Summary
The transgenic RNAi fly stock libraries (e.g., the Vienna Drosophila RNAi library[1] and the Transgenic RNAi Project (TRiP)) have been a tremendous boon to the Drosophila community because they permit tissue-specific knockdown of almost all genes in the genome. The relationship between a miRNA seed sequence and its complements in the open reading frames and 3′ -untranslated regions (3′ -UTRs) of target mRNAs spurred the development of bioinformatic algorithms that convert mature miRNA sequences into lists of potential mRNA targets[5] These lists of candidate targets, are plagued by large numbers of false positives because the algorithms that generate them can fully account for neither the precise spatial and temporal patterns of miRNA and target mRNA expression nor target site availability. MiRNA over-expression in arbitrary tissues using the binary GAL4/UAS expression system would likely lead to non-biological miRNA-target mRNA pairings Rather than seeing these pairings as a potential drawback of using a library of UAS-miRNA stocks, we expect they can be useful as part of a two-tiered screening system. The spatial arrangement of the 17 subclasses of adult olfactory sensilla on the antenna, the arrangement of the OSNs themselves, the precise pattern of OR expression, and the wiring of the antennal OSNs into the appropriate glomeruli of the antennal lobe are all highly stereotyped from fly to fly, indicating well-orchestrated developmental control of every step in the process
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