Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct;16(10):1016-20.
doi: 10.1038/nsmb.1675. Epub 2009 Aug 27.

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

Nicolas J Lehrbach  1 , Javier Armisen, Helen L Lightfoot, Kenneth J Murfitt, Anthony Bugaut, Shankar Balasubramanian, Eric A Miska
Affiliations

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.

PubMed Disclaimer

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 μg 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

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

Publication types

MeSH terms