Article | Published:

Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms

Nature Methods volume 6, pages 897903 (2009) | Download Citation

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.

Author information

Author notes

    • Carlos M Loya
    •  & Cecilia S Lu

    These authors contributed equally to this work.

Affiliations

  1. Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA.

    • Carlos M Loya
    • , Cecilia S Lu
    • , David Van Vactor
    •  & Tudor A Fulga

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Contributions

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.

Corresponding authors

Correspondence to David Van Vactor or Tudor A Fulga.

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DOI

https://doi.org/10.1038/nmeth.1402

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