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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References
Pasquinelli, A. E. et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408, 86–89 (2000).
Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906 (2000).
Houbaviy, H. B., Murray, M. F. & Sharp, P. A. Embryonic stem cell-specific microRNAs. Dev. Cell 5, 351–358 (2003).
Sempere, L. F. et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13 (2004).
Johnson, S. M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005).
Johnson, C. D. et al. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67, 7713–7722 (2007).
Ibarra, I., Erlich, Y., Muthuswamy, S. K., Sachidanandam, R. & Hannon, G. J. A role for microRNAs in maintenance of mouse mammary epithelial progenitor cells. Genes Dev. 21, 3238–3243 (2007).
Yu, F. et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109–1123 (2007).
Slack, F. J. et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, 659–669 (2000).
Meroni, G. & Diez-Roux, G. TRIM/RBCC, a novel class of 'single protein RING finger' E3 ubiquitin ligases. Bioessays 27, 1147–1157 (2005).
Slack, F. J. & Ruvkun, G. A novel repeat domain that is often associated with RING finger and B- box motifs. Trends Biochem. Sci. 23, 474–475 (1998).
Edwards, T. A., Wilkinson, B. D., Wharton, R. P. & Aggarwal, A. K. Model of the brain tumor-Pumilio translation repressor complex. Genes Dev. 17, 2508–2513 (2003).
Schulman, B. R., Esquela-Kerscher, A. & Slack, F. J. Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis. Dev. Dyn. 234, 1046–1054 (2005).
Lancman, J. J. et al. Analysis of the regulation of lin-41 during chick and mouse limb development. Dev. Dyn. 234, 948–960 (2005).
Kanamoto, T., Terada, K., Yoshikawa, H. & Furukawa, T. Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans. Dev. Dyn. 235, 1142–1149 (2006).
Maller Schulman, B. R. et al. The let-7 microRNA target gene, mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle 7, 3935–3942 (2008).
Wulczyn, F. G. et al. Post-transcriptional regulation of the let-7 microRNA during neural cell specification. FASEB J. 21, 415–426 (2007).
Rybak, A. et al. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nature Cell Biol. 10, 987–993 (2008).
Trabucchi, M. et al. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 459, 1010–1014 (2009).
Suzuki, H. I. et al. Modulation of microRNA processing by p53. Nature 460, 529–533 (2009).
Lee, Y. S., Kim, H. K., Chung, S., Kim, K. S. & Dutta, A. Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J. Biol. Chem. 280, 16635–16641 (2005).
Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).
Smirnova, L. et al. Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21, 1469–1477 (2005).
Xu, C., Zhou, Z. Y., Guo, Q. S. & Wang, Y. F. Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period. Asian J. Androl. 6, 217–222 (2004).
Yoshinaga, K. et al. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113, 689–699 (1991).
Kedersha, N. & Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61–81 (2007).
Duchaine, T. F. et al. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell 124, 343–354 (2006).
Balastik, M. et al. Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration. Proc. Natl Acad. Sci. USA 105, 12016–12021 (2008).
Diederichs, S. et al. Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. Proc. Natl Acad. Sci. USA 105, 9284–9289 (2008).
Diederichs, S. & Haber, D. A. Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression. Cell 131, 1097–1108 (2007).
Newman, M. A., Thomson, J. M. & Hammond, S. M. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA 14, 1539–1549 (2008).
Viswanathan, S. R., Daley, G. Q. & Gregory, R. I. Selective blockade of microRNA processing by Lin-28. Science 320, 97–100 (2008).
Heo, I. et al. Lin-28 mediates the terminal uridylation of let-7 precursor microRNA. Mol. Cell 32, 276–284 (2008).
Tokumaru, S., Suzuki, M., Yamada, H., Nagino, M. & Takahashi, T. let-7 regulates Dicer expression and constitutes a negative feedback loop. Carcinogenesis (2008).
Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008).
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).
Rodriguez, A. et al. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster. Genetics 143, 929–940 (1996).
Arama, E., Dickman, D., Kimchie, Z., Shearn, A. & Lev, Z. Mutations in the β-propeller domain of the Drosophila brain tumor (brat) protein induce neoplasm in the larval brain. Oncogene 19, 3706–3716 (2000).
Neumuller, R. A. et al. Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454, 241–245 (2008).
Locke, M., Tinsley, C. L., Benson, M. A. & Blake, D. J. TRIM32 is an E3 ubiquitin ligase for dysbindin. Hum. Mol. Genet. 18, 2344–2358 (2009).
Schwamborn, J. C., Berezikov, E. & Knoblich, J. A. The TRIM–NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136, 913–925 (2009).
Chang, T. C. et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nature Genet. 40, 43–50 (2008).
Loedige, I. & Filipowicz, W. TRIM–NHL proteins take on miRNA regulation. Cell 136, 818–820 (2009).
Hammell, C. M., Lubin, I., Boag, P. R., Blackwell, T. K. & Ambros, V. nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell 136, 926–938 (2009).
Parry, D. H., Xu, J. & Ruvkun, G. A whole-genome RNAi Screen for C. elegans miRNA pathway genes. Curr.Biol. 17, 2013–2022 (2007).
Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).
Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nature Cell Biol. 7, 1267–1274 (2005).
Makeyev, E. V., Zhang, J., Carrasco, M. A. & Maniatis, T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol.Cell 27, 435–448 (2007).
Oeckinghaus, A. et al. Malt1 ubiquitination triggers NF-κB signaling upon T-cell activation. EMBO J. 26, 4634–4645 (2007).
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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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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DOI: https://doi.org/10.1038/ncb1987
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