Abstract
The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and developmental timing and a candidate tumor suppressor. Here we show that LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 processing in Caenorhabditis elegans. We demonstrate that lin-28 is necessary and sufficient to block let-7 activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. Moreover, we have identified a poly(U) polymerase, PUP-2, which regulates the stability of LIN-28–blockaded let-7 pre-miRNA and contributes to LIN-28–dependent regulation of let-7 during development. We show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 pre-miRNA by PUP-2 in vitro. Our results demonstrate that LIN-28 and let-7 form an ancient regulatory switch, conserved from nematodes to humans, and provide insight into the mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 ortholog might regulate let-7 and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and cancer biology, we propose that such poly(U) polymerases are potential therapeutic targets.
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Acknowledgements
We thank A. Hutterer (Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge) for a strain carrying the mjIs15 transgene, M. Jackman (Wellcome Trust Cancer Research UK Gurdon Institute) for the pDEST-MAL, pcDNA5/FRT/TO_GATEWAY_TEV_SBP and pDEST-3Flag 3HA vectors, E. Moss (University of Medicine and Dentistry of New Jersey, USA) for anti–LIN-28 antibody and M. Wickens (University of Wisconsin, USA) for PUP-2 cDNA. We thank R. Gregory and N. Kim for sharing unpublished data. N.J.L. and K.J.M. were supported by a PhD studentship from the Wellcome Trust. J.A. and A.B. were supported by grants from the Biotechnology and Biological Sciences Research Council (UK). H.L.L. was supported by a PhD studentship from Cancer Research UK. This work was supported by Cancer Research UK Programme Grants to E.A.M. and S.B. and core funding to the Wellcome Trust/Cancer Research UK Gurdon Institute provided by the Wellcome Trust and Cancer Research UK.
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N.J.L., S.B. and E.A.M. conceived the original project; N.J.L. carried out all experiments unless stated otherwise; J.A. carried out northern blotting, uridylase assays and microarray experiments; H.L.L., J.A. and A.B. carried out RNA mobility shift assays; K.J.M. and J.A. carried out some of the in vitro binding assays; N.J.L., J.A. and E.A.M. wrote the manuscript.
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Lehrbach, N., Armisen, J., Lightfoot, H. et al. LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans. Nat Struct Mol Biol 16, 1016–1020 (2009). https://doi.org/10.1038/nsmb.1675
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DOI: https://doi.org/10.1038/nsmb.1675
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