LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans - PubMed (original) (raw)

. 2009 Oct;16(10):1016-20.

doi: 10.1038/nsmb.1675. Epub 2009 Aug 27.

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LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans

Nicolas J Lehrbach et al. Nat Struct Mol Biol. 2009 Oct.

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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Figures

Figure 1

Figure 1. A quantitative assay reveals post-transcriptional regulation of the let-7 miRNA by lin-28

(a,b) Schematic of the pharynx based assay of let-7 activity. (c) Fluorescence images of animals carrying the let-7 sensor transgene at L1 larval and adult stages. Both GFP and mCherry are strongly expressed. d Fluorescence images of animals carrying both the let-7 sensor and myo-2::let-7 transgenes at L1 larval and adult stages. GFP is specifically and robustly downregulated in adults, but not in L1 larvae. Scale bar shows 20 μm. e Fluorescence images showing that lin-28 mutants downregulate let-7 sensor GFP at the L1 stage in a myo-2::let-7 dependent fashion. This effect is not reversed in a lin-46 mutant background. Scale bar shows 20 μm. f Fluorescence images showing that a myo-2::lin-28::unc-54 transgene is sufficient to block let-7 activity in adults carrying let-7 sensor and myo-2::let-7 transgenes. Scale bar shows 20 μm.

Figure 2

Figure 2. pup-2 regulates let-7 processing in a _lin-28_-dependent fashion

a let-7 is uridylated in vivo. Frequency of unmodified and modified let-7* molecules identified by high-throughput sequencing. b Representative northern blot showing _pup-2_-dependent regulation of pre-let-7. 5 _μ_g of total RNA from control, lin-28(RNAi), and pup-2(RNAi) myo-2::let-7 L2 larvae was loaded. U6 was used as a loading control. c,d Quantification of relative pre-let-7 (c), and let-7 (d) abundance in lin-28(RNAi), pup-2(RNAi) and cid-1(RNAi) myo-2::let-7 L2 and L4 larvae from northern blotting experiments. Mean fold change relative to empty vector control samples is shown. _P_-values from Students’ _t_-tests indicated; n = 4. Error bars show standard error of the mean. e Fluorescence image showing the seam cell defect observed in pup-2(RNAi) adults. A DLG-1-mCherry fusion marks seam cell boundaries. Upper panel; wild-type with continuous seam. Lower panel; pup-2(RNAi) with incompletely fused seam. Arrows indicate sites of failed fusion. Scale bar shows 20 μm.

Figure 3

Figure 3. LIN-28 interacts with PUP-2 and promotes uridylation of pre-let-7 by PUP-2

a Co-Immunoprecipitation of PUP-2 and LIN-28 expressed in HEK293T cells. b GST pull-down assay demonstrating a direct interaction of GST-LIN-28 and PUP-2 in vitro. c In vitro uridylation assay showing that PUP-2 uridylates pre-let-7 in a LIN-28 dependent fashion.

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References

    1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
    1. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. - PMC - PubMed
    1. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009;10:126–139. - PubMed
    1. Ambros V, Horvitz HR. Heterochronic mutants of the nematode Caenorhabditis elegans. Science. 1984;226:409–416. - PubMed
    1. Chalfie M, Horvitz HR, Sulston JE. Mutations that lead to reiterations in the cell lineages of C. elegans. Cell. 1981;24:59–69. - PubMed

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