Abstract
MicroRNAs (miRNAs) regulate gene expression by pairing with complementary sequences in the 3′ untranslated regions (UTRs) of transcripts. Although the molecular mechanism underlying miRNA biogenesis and activity is becoming better understood, determining the physiological role of the interaction of an miRNA with its target remains a challenge. A number of methods have been developed to inhibit individual miRNAs, but it can be difficult to determine which specific targets are responsible for any observed phenotypes. To address this problem, we use target protector (TP) morpholinos that interfere with a single miRNA-mRNA pair by binding specifically to the miRNA target sequence in the 3′ UTR. In this protocol, we detail the steps for identifying miRNA targets, validating their regulation and using TPs to interrogate their function in zebrafish. Depending on the biological context, this set of experiments can be completed in 6–8 weeks.
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Acknowledgements
We acknowledge W.-Y. Choi and A. Schier, who together with A.J.G., developed the target protector technology. This work was supported by a National Research Service Award–US National institutes of Health (NIH)/National Institute of General Medical Sciences T32 GM007223 Training Grant (A.A.S.); and by NIH grants R01GM081602-05, the Yale Scholar program and the Pew Scholars Program in Biomedical Sciences (A.J.G.).
AUTHOR CONTRIBUTIONS
A.A.S. and A.J.G. designed experiments and interpreted results. A.A.S. performed all experiments and prepared the manuscript with input from A.J.G.
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A.J.G. is an author on a patent to use TP technology to block the regulation of target mRNAs.
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Staton, A., Giraldez, A. Use of target protector morpholinos to analyze the physiological roles of specific miRNA-mRNA pairs in vivo. Nat Protoc 6, 2035–2049 (2011). https://doi.org/10.1038/nprot.2011.423
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DOI: https://doi.org/10.1038/nprot.2011.423
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