Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28

Nature Structural & Molecular Biology volume 19, pages 84–89 (2012)Cite this article

Subjects

Abstract

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5′-AGGAGAU-3′. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5′-NGNNG-3′ consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The ZnFs of Lin28 bind single-stranded regions of pre-let-7 terminal loops.
Figure 2: The solution structure of Lin28 ZnF12 bound to 5′-AGGAGAU-3′.
Figure 3: Affinity of Lin28 variants for single-stranded and pre-let-7g RNAs and processing of pre-let-7g point mutants in Huh-7 cells.
Figure 4: Comparison between the structure of Lin28 ZnFs bound to 5′-AGGAGAU-3′ (this study) and HIV nucleocapsid (NC) bound to stem-loops of the RNA packaging signal.

Similar content being viewed by others

Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Bushati, N. & Cohen, S.M. microRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Krol, J., Loedige, I. & Filipowicz, W. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet. 11, 597–610 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Viswanathan, S.R. & Daley, G.Q. Lin28: a microRNA regulator with a macro role. Cell 140, 445–449 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Büssing, I., Slack, F.J. & Grosshans, H. let-7 microRNAs in development, stem cells and cancer. Trends Mol. Med. 14, 400–409 (2008).

    Article  PubMed  Google Scholar 

  5. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Viswanathan, S.R., Daley, G.Q. & Gregory, R.I. Selective blockade of microRNA processing by Lin28. Science 320, 97–100 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Van Wynsberghe, P.M. et al. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 18, 302–308 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lightfoot, H.L. et al. A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing. Biochemistry 50, 7514–7521 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Rybak, A. et al. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat. Cell Biol. 10, 987–993 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Hagan, J.P., Piskounova, E. & Gregory, R.I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Heo, I. et al. Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol. Cell 32, 276–284 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Lehrbach, N.J. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Xu, B. & Huang, Y. Histone H2a mRNA interacts with Lin28 and contains a Lin28-dependent posttranscriptional regulatory element. Nucleic Acids Res. 37, 4256–4263 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Qiu, C., Ma, Y., Wang, J., Peng, S. & Huang, Y. Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells. Nucleic Acids Res. 38, 1240–1248 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Balzer, E., Heine, C., Jiang, Q., Lee, V.M. & Moss, E.G. LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro. Development 137, 891–900 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Jin, J. et al. Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes. Nucleic Acids Res. 39, 3724–3734 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Viswanathan, S.R. et al. Lin28 promotes transformation and is associated with advanced human malignancies. Nat. Genet. 41, 843–848 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lu, K., Heng, X. & Summers, M.F. Structural determinants and mechanism of HIV-1 genome packaging. J. Mol. Biol. 410, 609–633 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Piskounova, E. et al. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J. Biol. Chem. 283, 21310–21314 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Desjardins, A., Yang, A., Bouvette, J., Omichinski, J.G. & Legault, P. Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28. Nucleic Acids Res. 10.1093/nar/gkr808 (2011).

  23. Summers, M.F. et al. Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1. Protein Sci. 1, 563–574 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yeom, K.H. et al. Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation. EMBO Rep. 12, 690–696 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. South, T.L. & Summers, M.F. Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC. Protein Sci. 2, 3–19 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. De Guzman, R.N. et al. Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279, 384–388 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Amarasinghe, G.K. et al. NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. J. Mol. Biol. 301, 491–511 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. D'Souza, V. & Summers, M.F. Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus. Nature 431, 586–590 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Duss, O., Maris, C., von Schroetter, C. & Allain, F.H. A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage. Nucleic Acids Res. 38, e188 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).

    Article  CAS  Google Scholar 

  33. Pelton, J.G., Torchia, D.A., Meadow, N.D. & Roseman, S. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques. Protein Sci. 2, 543–558 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Schubert, M., Manolikas, T., Rogowski, M. & Meier, B.H. Solid-state NMR spectroscopy of 10% 13C labeled ubiquitin: spectral simplification and stereospecific assignment of isopropyl groups. J. Biomol. NMR 35, 167–173 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Dominguez, C., Schubert, M., Duss, O., Ravindranathan, S. & Allain, F.H. Structure determination and dynamics of protein-RNA complexes by NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 58, 1–61 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Lee, W., Revington, M.J., Arrowsmith, C. & Kay, L.E. A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes. FEBS Lett. 350, 87–90 (1994).

    Article  CAS  PubMed  Google Scholar 

  37. Peterson, R.D., Theimer, C.A., Wu, H. & Feigon, J. New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes. J. Biomol. NMR 28, 59–67 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Iwahara, J., Wojciak, J.M. & Clubb, R.T. Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment. J. Biomol. NMR 19, 231–241 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Herrmann, T., Guntert, P. & Wuthrich, K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ludvigsen, S. & Poulsen, F.M. Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy. J. Biomol. NMR 2, 227–233 (1992).

    Article  CAS  PubMed  Google Scholar 

  42. Schwabe, J.W. & Klug, A. Zinc mining for protein domains. Nat. Struct. Biol. 1, 345–349 (1994).

    Article  CAS  PubMed  Google Scholar 

  43. Legge, G.B. et al. ZZ domain of CBP: an unusual zinc finger fold in a protein interaction module. J. Mol. Biol. 343, 1081–1093 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Neuhaus, D., Nakaseko, Y., Schwabe, J.W. & Klug, A. Solution structures of two zinc-finger domains from SWI5 obtained using two-dimensional 1H nuclear magnetic resonance spectroscopy. A zinc-finger structure with a third strand of beta-sheet. J. Mol. Biol. 228, 637–651 (1992).

    Article  CAS  PubMed  Google Scholar 

  45. Case, D.A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668–1688 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pang, Y.P., Xu, K., Yazal, J.E. & Prendergas, F.G. Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach. Protein Sci. 9, 1857–1865 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Blatter for assistance in the structure calculations, M. Zimmermann for synthesis of DNA templates, M. Roos for cell culture preparations and P. Wenter (Eurofins MWG Operon) for synthesis of biotinylated RNAs. This work was supported by an Eidgenössische Technische Hochschule (ETH) postdoctoral fellowship to F.E.L., Sinergia grant CRSII3_127454 from the Swiss National Science Foundation (SNF) to F.H.-T.A. and J.H., Krebsforschung Schweiz (KFS) grant 02648-08-2010 to F.H.-T.A. and J.H., and SNF grant 205321_124720 to J.H.

Author information

Authors and Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland

    Fionna E Loughlin & Frédéric H-T Allain

  2. Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland

    Luca F R Gebert, Harry Towbin, Andreas Brunschweiger & Jonathan Hall

Authors
  1. Fionna E Loughlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luca F R Gebert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Harry Towbin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andreas Brunschweiger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonathan Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frédéric H-T Allain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.H.-T.A., F.E.L. and J.H. designed the project; F.E.L. prepared protein and RNA samples for structural studies; F.E.L. and F.H.-T.A. collected and analyzed NMR data; F.E.L. carried out the structure calculations and the ITC measurements; H.T. and A.B. did the Lin28 binding assay with miRNAs and L.F.R.G. did the quantitative PCR in cell assays. All authors discussed the results, wrote and approved the manuscript.

Corresponding authors

Correspondence to Jonathan Hall or Frédéric H-T Allain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 7283 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Loughlin, F., Gebert, L., Towbin, H. et al. Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28. Nat Struct Mol Biol 19, 84–89 (2012). https://doi.org/10.1038/nsmb.2202

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2202

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing