Abstract
Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and likely proto-oncogenes, selectively inhibit the maturation of let-7 family microRNAs (miRNAs) in embryonic stem cells and certain cancer cell lines. Moreover, let-7 precursors (pre–let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3′ terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre–let-7 uridylation and subsequent blockade of let-7 processing in mouse embryonic stem cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7 and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a role for Zcchc11-catalyzed pre–let-7 uridylation in the control of miRNA biogenesis.
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Acknowledgements
We are grateful to S. Viswanathan and G. Daley (Children's Hospital Boston) for providing Flag-Lin28–expressing ES cells, H. Rusk and R. LaPierre for technical assistance and the Proteomics Center at Children's Hospital Boston for expertise in the microcapillary HPLC and MS. We thank E. Miska (University of Cambridge) for helpful discussions. R.I.G. is supported by laboratory start-up funds from The Children's Hospital Boston and grants from the US National Institute of General Medical Sciences (NIGMS) (1R01GM086386-01A1), The Harvard Stem Cell Institute, The March of Dimes Basil O'Conner award and the Emerald Foundation. R.I.G. is supported by The Pew Scholars Program in the Biomedical Sciences.
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J.P.H. and E.P. performed all experiments; J.P.H., E.P. and R.I.G. designed all experiments, analyzed data and wrote the manuscript.
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Hagan, J., Piskounova, E. & Gregory, R. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat Struct Mol Biol 16, 1021–1025 (2009). https://doi.org/10.1038/nsmb.1676
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DOI: https://doi.org/10.1038/nsmb.1676
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