Abstract
MicroRNAs are important regulators of gene expression, yet the functional outputs of most microRNA-target interactions remain elusive. Here we introduce the Drosophila melanogaster microRNA sponge (miR-SP) as a powerful transgenic technology to dissect the function of every microRNA with precise spatiotemporal resolution. miR-SPs can be used to characterize tissue-specific microRNA loss-of-function phenotypes, define the spatial regulation of their effectors and uncover interactions between microRNAs and other genes. Using themiR-SP system, we identified an essential role of the conserved microRNA miR-8, in neuromuscular junction formation. Tissue-specific silencing revealed that postsynaptic activity of miR-8 is important for normal neuromuscular junction morphogenesis. Given that miR-SPs rely on a bipartite modular expression system, they could be used to elucidate the endogenous function of microRNAs in any species in which conditional expression can be achieved.
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Acknowledgements
We thank M. Greenberg, N. Perrimon, T. Schwarz, M. Feany and J. Flanagan for critical feedback on the manuscript; M. Ebert for advice on designing the miR-SP constructs and for comments on the manuscript; V. Sridhar and members of the Harvard NeuroDiscovery Center Optical Imaging program for technical assistance; R.W. Carthew (Northwestern University) for GMR-YanAct; F.B. Gao (University of California San Francisco) for miR-9aJ22 and miR-9aE39; M. Peifer (University of North Carolina) for UAS-FP4-mitoEGFP; F.M. Hoffmann (University of Wisconsin-Madison) for ena210 and UAS-ena; H. Ruohola-Baker (University of Washington) for miR-8-GFP sensor; J. Brennecke (Institutes of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna) for nerfin-1 3′ UTR reporter; S. Cohen (Temasek Life Sciences Laboratory) for miR-8Δ2; and members of the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biological Sciences) for antibodies. C.M.L., C.S.L. and D.V.V. were supported by a grant from the National Institutes of Health (NS40043). T.A.F. was supported in part by a fellowship from the John Douglas French Alzheimer's Foundation.
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T.A.F. conceived and designed the miR-SP technology. C.M.L. and T.A.F. designed and performed the experiments, analyzed the data and interpreted the results. C.S.L. generated the miR-8 null mutant and independent of this study performed preliminary NMJ analysis and proposed Ena as a candidate miR-8 effector. D.V.V. supervised the study and provided material and salary support for C.M.L., C.S.L. and T.A.F. The text and figures were drafted by T.A.F., and T.A.F., D.V.V. and C.M.L. edited them.
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Loya, C., Lu, C., Van Vactor, D. et al. Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms. Nat Methods 6, 897–903 (2009). https://doi.org/10.1038/nmeth.1402
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DOI: https://doi.org/10.1038/nmeth.1402
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