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The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2

Abstract

The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.

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Figure 1: Regulatory interactions between mLin41 and let-7 (a) Ectopic let-7 and miR-125 decrease mLin41 mRNA in EC cells.
Figure 2: mLin41 expression in mouse epiblasts and embryonic ectoderm.
Figure 3: mLin41 expression in male germ cells.
Figure 4: mLin41 localizes to P-bodies.
Figure 5: mLin41 interacts with Ago2 in co-precipitation assays.
Figure 6: mLin41 is an E3 ubiquitin ligase for Ago2.
Figure 7: mLin41 antagonizes Ago2 and cooperates with lin-28.

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Acknowledgements

The authors would like to thank members of the Robert Nitsch laboratories, in particular Vincenzo Catanzariti for assistance with figure preparation, and Ari Liebkowsky, Elisa Cuevas, Eleonora Franzoni and Junko Toraiwa for critical reading of the manuscript. Christian Hagemeier kindly provided tissue-specific cDNAs used in preliminary work. Michele Salanova (testis) and Sven Hendrix and Ruth Schmidt-Ulrich (skin) helped with methods and interpretation of immunohistochemistry results. Nicola Brandt and Kristin Franke generously provided cultured hippocampal neurons. We acknowledge the laboratory support of Brita Scholte. L.S. and A.R. were fellows of the Humboldt University Graduate Schools 238 and 1123, respectively. Additional support was provided by SFB grant 665 to F.G.W. and R.N.

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A. R., H. F., K. H., L. S., D. K. and F. G. W. contributed to experimental planning and design; A. R., H. F., K. H., L. S. and E. A. W. performed experimental work; G. M. supervised early embryo experiments; R. N. provided intellectual and supervisory support.

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Correspondence to F. Gregory Wulczyn.

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Rybak, A., Fuchs, H., Hadian, K. et al. The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nat Cell Biol 11, 1411–1420 (2009). https://doi.org/10.1038/ncb1987

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