The in vivo genetic toolkit for studying expression and functions of Drosophila melanogaster microRNAs

Abstract

Since the initial reports that a group of small RNAs, now known as microRNAs (miRNAs), regulates gene expression without being translated into proteins, there has been an explosion of studies on these important expression modulators. Drosophila melanogaster has proven to be one of the most amenable animal models for investigations of miRNA biogenesis and gene regulatory activities. Here, we highlight the publicly available genetic tools and strategies for in vivo functional studies of miRNA activity in D. melanogaster. By coupling genetic approaches using available strain libraries with technologies for miRNA expression analysis and target and pathway prediction, researchers' ability to test functional activities of miRNAs in vivo is now greatly enhanced. We also comment on the tools that need to be developed to aid in comprehensive evaluation of Drosophila miRNA activities that impact traits of interest.

Keywords: Drosophila melanogaster; genetics; in vivo; microRNA; tools.

Figure 1.

miRNA tools for in vivo studies. (A) Targeted deletions are available at public stock centers for 130 out of 256 miRNA loci in Drosophila. The loci were deleted either singly or in clusters. (B) UAS-miRNA sponges are available for reducing the activity of 141 independent miRNAs in specific tissues or cells. (C) UAS-miRNA expression constructs for use in misexpression, overexpression and mutant rescue experiments are also available for a subset of miRNAs. A, B, C and D are generic designations for Drosophila miRNAs.

Figure 2.

Variations of the GAL4/UAS system in common use. (A) The GAL4/UAS system can be used to manipulate gene expression either ubiquitously or in a tissue-specific manner (illustrated in thoracic muscle as an example). Crosses between a strain carrying enhancer-gal4 (ubiquitous or tissue-specific enhancer) and a strain with the UAS-gene-of-interest (gene coding sequence, RNAi, miRNA-sponge, etc. represented in blue) produce flies containing both elements. In enhancer-gal4 parents and progeny, GAL4 is always expressed based on the activity of the enhancer (represented by yellow areas). In progeny from the cross, the UAS-gene-of-interest will be activated where GAL4 is present, either ubiquitously or restricted to the same tissue-specific regions as the enhancer (represented by green in the figure). (B) Temporal control of GAL4/UAS activity can be achieved by repression of GAL4 by Gal80^ts which restricts activity at low temperatures (represented by gray areas) but is inactivated at higher temperatures, thereby allowing widespread expression of the gene of interest (green body). (C) Both temporal and spatial control of gene expression can be achieved by combining a tissue-specific enhancer-gal4 with Gal80^ts (green thorax).