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Biological basis for restriction of microRNA targets to the 3′ untranslated region in mammalian mRNAs

Nature Structural & Molecular Biology volume 16, pages 144–150 (2009)Cite this article

Abstract

MicroRNAs (miRNAs) interact with target sites located in the 3′ untranslated regions (3′ UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3′ UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

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Figure 1: miRNA-mediated repression is abolished in extended ORFs.
Figure 2: miRNA-mediated repression studies were concordant in mouse liver in vivo.
Figure 3: Insertion of rare codons upstream of the extended miRNA ORF rescues miRNA-mediated knockdown.
Figure 4: Insertion of rare codons increases the accessibility of downstream sequences to RNase H–mediated cleavage.

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Acknowledgements

This work was supported by the US National Institutes of Health grant DK 78424. We thank B. Hu for helping prepare some of the samples, R. Cevailos for technical assistance with the polyribosome fractionation experiments and D. Haussecker for critical reading of the manuscript.

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Authors and Affiliations

  1. The Center for Clinical Science Research, Room 2105, 269 Campus Drive, Stanford, California 94305-5164, USA.,

    Shuo Gu, Lan Jin, Feijie Zhang & Mark A Kay

  2. Department of Microbiology and Immunology, 300 Pasteur Drive, Room D309, Stanford University, Stanford, 94305, California, USA

    Peter Sarnow

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  1. Shuo Gu
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Contributions

S.G. designed and implemented most of the experiments; L.J. performed the studies outlined in Figure 4; F.Z. assisted S.G. with the molecular biology preparations; P.S. provided assistance with the polysome studies and offered critical discussions related to data interpretation; M.A.K. supervised the studies and provided scientific input into the experimental design and data interpretation; S.G and M.A.K wrote the manuscript; all authors approved the final manuscript.

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Correspondence to Mark A Kay.

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Gu, S., Jin, L., Zhang, F. et al. Biological basis for restriction of microRNA targets to the 3′ untranslated region in mammalian mRNAs. Nat Struct Mol Biol 16, 144–150 (2009). https://doi.org/10.1038/nsmb.1552

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