A genome-wide transgenic resource for conditional expression ofDrosophilamicroRNAs

Abstract

SUMMARYmicroRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila

“…In general, while our previous survey of well-conserved Drosophila miRNAs showed that the vast majority (>80%) could induce mutant phenotypes when ectopically expressed (Bejarano et al 2012), most recently evolved miRNAs did not share this capacity. However, by segregating newly evolved miRNAs according to whether they were testis restricted or not, we observed that the former set exhibit dramatically greater phenotypic capacity (Fig.…”

“…We expanded our recently published collection of conditional UAS-DsRedmiRNA expression transgenes (Bejarano et al 2012) by generating 28 additional transgenic lines (Supplemental Tables 4, 5), mostly composed of newly evolved miRNAs that we had annotated more recently (Berezikov et al 2011;Chung et al 2011). We then selected all the transgenes for newly evolved miRNAs and performed a systematic screen of all the independent insertions for each construct against nine Gal4 drivers, including ubiquitous (da-Gal4), eye specific (GMR-Gal4, ey-Gal4), wing specific (Sd-Gal4), wing and notum (1096-Gal4), anterior posterior compartment boundary (dpp-Gal4 and ptc-Gal4), notum specific (Eq-Gal4), and neuron specific (elav-Gal4).…”

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle

“…Thus, it may be necessary to perform a genetic screen of individual miRNAs in fly head circadian cells to investigate their potential role in circadian regulation. Recently, a transgenic UAS-miRNA library in Drosophila has been constructed [54][55][56] . This library circumvents the redundancy issues inherent in loss-of-function screens by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches.…”

Functional Characterization of Drosophila microRNAs by a Novel in Vivo Library

“…This in vivo library provides an important genetic tool that complements miRNA loss-of-function approaches (e.g., knock-outs, deletions, and miRNA sponges). While this work was under review, Lai and colleagues described their efforts to generate a similar UAS-miRNA resource (Bejarano et al 2012). Furthermore, an earlier publication reported a smaller collection of UAS-miRNA lines that was used as a tool to complement genetic deficiency screening (Szuplewski et al 2012).…”

“…The abundance of the mature miRNA depends only on the processing efficiency of the hairpin precursor by the endogenous biogenesis machinery. Importantly, in contrast to Bejarano et al (2012), our cloning system focuses only on the pre-miRNA hairpin without adding additional sequences or markers. All our constructs were cloned into identical vectors.…”