An accelerated miRNA-based screen implicates Atf-3 in Drosophila odorant receptor expression

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. To mitigate some of the challenges of genome-wide screening, we propose 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 target mRNAs, miRNA over-expression phenotypes should be attributable to target loss-of-function. Since miRNA-target pairing is sequence-dependent, predicted targets of miRNAs identified in the primary screen are 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 a proof-of-concept miRNA-based screen that uncovers a role for Atf3 in the expression of the odorant receptor Or47b.

Introduction

The transgenic RNAi fly stock libraries (e.g., the Vienna Drosophila RNAi library (Dietzl 2007) 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. These resources permit genome-wide screens for genes associated with almost any phenotype of interest. Unfortunately, the sheer size of these libraries—more than 22,000 stocks in the case of the Vienna library—means performing such screens remains labor-intensive and tedious. In this paper, we describe our development of a two-tiered screening protocol comprising an initial pooling screen using miRNA over-expression that generates a list of candidate genes involved in a phenotype of interest and a secondary screen using gene-specific RNAi that narrows this list of candidates to the responsible target gene(s). We suggest that this protocol can sometimes accelerate the identification of novel genes involved in a broad range of phenotypes.

MicroRNAs are short, endogenous, single-stranded RNA molecules that act in the context of the miRISC protein complex to either inhibit translation or induce the degradation of target mRNAs (Bartel 2004). Since the miRNA-target mRNA relationship is determined primarily by a short seed sequence at the 5’ end of each miRNA (Lewis 2003, Lai 2002), the complement of which may occur in multiple copies scattered over the genome, many miRNAs are capable of down-regulating multiple targets. 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 (Rajewsky 2006). These lists of candidate targets, however, 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. In other words, a miRNA may be capable of down-regulating a particular target and never actually do so, either because the two are never simultaneously expressed in the same tissue or because RNA-binding proteins or RNA folding render the target site inaccessible. It also follows that miRNA over-expression in arbitrary tissues using the binary GAL4/UAS expression