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Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

Nature Structural & Molecular Biology volume 17, pages 173–179 (2010)Cite this article

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Abstract

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C. elegans. Using cross-linking and ALG-1 immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), we identified extensive ALG-1 interactions with specific 3′ untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3′ UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.

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Figure 1: MicroRNA targets identified by ALG-1 CLIP-seq in L4-stage worms.
Figure 2: Relative position of ALG-1 binding sites across protein-coding genes.
Figure 3: Attributes enriched in ALG-1 binding sites within 3′ UTRs.
Figure 4: Relationship between ALG-1 binding and mRNA expression levels.

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Acknowledgements

The authors thank G. Ruvkun, X. Fu, W. McGinnis and members of our laboratories for critical reading of the manuscript. We thank B. Hehli, S. Hunter and S. Bagga for technical assistance, V. Ambros and M. Hammell for providing the list of mirWIP predictions and D. Bartel for helpful advice. MLW is supported by the Genetics Training Program at the University of California, San Diego and a graduate fellowship from Genentech. This work was supported by grants from the US National Institutes of Health (GM071654-01 to A.E.P. and HG004659 and GM084317 to G.W.Y.), the Keck, Searle, V., Emerald and Peter Gruber Foundations (A.E.P.) and the Stem Cell Program at the University of California, San Diego (G.W.Y.).

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  1. Dimitrios G Zisoulis and Michael T Lovci: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.,

    Dimitrios G Zisoulis & Amy E Pasquinelli

  2. Department of Cellular and Molecular Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.,

    Michael T Lovci, Melissa L Wilbert, Kasey R Hutt, Tiffany Y Liang & Gene W Yeo

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  1. Dimitrios G Zisoulis
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Contributions

A.E.P. and G.W.Y. designed and directed the project; A.E.P., D.G.Z., G.W.Y. and M.T.L. wrote the paper; D.G.Z. and T.Y.L. performed the experiments; M.T.L., M.L.W., K.R.H, T.Y.L. and G.W.Y. performed the bioinformatics analyses.

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Correspondence to Amy E Pasquinelli or Gene W Yeo.

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Zisoulis, D., Lovci, M., Wilbert, M. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat Struct Mol Biol 17, 173–179 (2010). https://doi.org/10.1038/nsmb.1745

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