Academic journal article Genetics

MicroRNA Transgene Overexpression Complements Deficiency-Based Modifier Screens in Drosophila

Academic journal article Genetics

MicroRNA Transgene Overexpression Complements Deficiency-Based Modifier Screens in Drosophila

Article excerpt

ABSTRACT Dosage-sensitive modifier screening is a powerful tool for linking genes to biological processes. Use of chromosomal deletions permits sampling the effects of removing groups of genes related by position on the chromosome. Here, we explore the use of inducible microRNA transgenes as a complement to deficiency-based modifier screens. miRNAs are predicted to have hundreds of targets. miRNA overexpression provides an efficient means to reduces expression of large gene sets. A collection of transgenes was prepared to allow overexpression of 89 miRNAs or miRNA clusters. These transgenes and a set of genomic deficiencies were screened for their ability to modify the bristle phenotype of the cell-cycle regulator minus. Sixteen miRNAs were identified as dominant suppressors, while the deficiency screen uncovered four genomic regions that contain a dominant suppressor. Comparing the genes uncovered by the deletions with predicted miRNA targets uncovered a small set of candidate suppressors. Two candidates were identified as suppressors of the minus phenotype, Cullin-4 and CG5199/Cut8. Additionally, we show that Cullin-4 acts through its substrate receptor Cdt2 to suppress the minus phenotype. We suggest that inducible microRNA transgenes are a useful complement to deficiency-based modifier screens.

GENETIC modifier screens have proven to be a powerful means with which to identify genes in a common biological process. Modifier screens can be carried out with high efficiency by chemical mutagenesis, but recombination mapping to identify the affected loci can be tedious. In Drosophila, a popular alternative has been to use a collection of deletion mutant strains known as the "deficiency kit" (St Johnston 2002), which consists of mapped chromosomal deletions that remove large blocks of genes. This approach allows sampling of the effects of removing one copy each of hundreds of genes in a single genetic cross. The deficiency kit currently available from the Bloomington Drosophila Stock Center covers 97.8% of annotated euchromatic genes (Cook et al. 2010). The advantage of the approach is that screens for autosomal modifiers can be performed with fewer than 400 crosses. An offsetting disadvantage is that genetic heterogeneity in these strains can affect the results.

In this context we decided to explore the possibility of using microRNAs (miRNAs) as an alternative means of downregulating hundreds of genes concurrently. miRNAs are small noncoding RNAs that post-transcriptionally silence gene expression. Target prediction algorithms suggest the existence of hundreds of targets per miRNA (reviewed in Bartel 2009; Thomas et al. 2010), recently including sites in protein-coding regions of the genes (Schnall-Levin et al. 2010). miRNA overexpression can cause simultaneous reduction of the expression levels of hundreds of genes (Lim et al. 2005; Easow et al. 2007; Baek et al. 2008; Selbach et al. 2008). Potential advantages of the use of miRNAs include the following:

1. miRNA targets may allow access to genes not covered by the deficiency kit, as well as facilitating access to genes on the X chromosome. There are 176 annotated Drosophila miRNAs (Kozomara and Griffiths-Jones 2011).

2. Overexpression studies can be conducted in a spatiotemporally controlled manner through the use of the GAL4-UAS system (Brand and Perrimon 1993). Potential drawbacks of the miRNA approach include: (a) miRNAs do not target all protein coding genes and (b) miRNAs do not regulate those they target with equal efficiency.

3. Identification of biologically significant targets is imperfect due to limitations in target prediction algorithms.

To explore the potential of the miRNA-based screening approach we made use of the minus mutant, which exhibits a "small bristle" phenotype that is sensitive to genetic background (Szuplewski et al. 2009). Drosophila adult mechanosensory bristles have been used as a model system to study cell determination and asymmetric cell division (Abdelilah- Seyfried et al. …

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