Article | Published:

Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis

Nature Structural & Molecular Biology volume 17, pages 10111018 (2010) | Download Citation

Abstract

The pluripotency-promoting proteins Lin28a and Lin28b act as post-transcriptional repressors of let-7 miRNA biogenesis in undifferentiated embryonic stem cells. The levels of mature let-7a differ substantially in cells lacking Lin28 expression, indicating the existence of additional mechanism(s) of post-transcriptional regulation. Here, we present evidence supporting a role for heteronuclear ribonucleoprotein A1 (hnRNP A1) as a negative regulator of let-7a. HnRNP A1 binds the conserved terminal loop of pri-let-7a-1 and inhibits its processing by Drosha. Levels of mature let-7a negatively correlate with hnRNP A1 levels in somatic cell lines. Furthermore, hnRNP A1 depletion increased pri-let-7a-1 processing by cell extracts, whereas its ectopic expression decreased let-7a production in vivo. Finally, hnRNP A1 binding to let-7a interferes with the binding of KSRP, which is known to promote let-7a biogenesis. We propose that hnRNP A1 and KSRP have antagonistic roles in the post-transcriptional regulation of let-7a expression.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

  2. 2.

    , , , & Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39, 673–677 (2007).

  3. 3.

    et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

  4. 4.

    , & Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 24, 138–148 (2005).

  5. 5.

    , & Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139 (2009).

  6. 6.

    , , , & Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region. Biochem. Biophys. Res. Commun. 304, 184–190 (2003).

  7. 7.

    , & The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14, 2162–2167 (2004).

  8. 8.

    , , , & Processing of primary microRNAs by the Microprocessor complex. Nature 432, 231–235 (2004).

  9. 9.

    et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

  10. 10.

    & Processing of intronic microRNAs. EMBO J. 26, 775–783 (2007).

  11. 11.

    et al. Primary microRNA transcripts are processed co-transcriptionally. Nat. Struct. Mol. Biol. 15, 902–909 (2008).

  12. 12.

    & Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production. J. Cell Biol. 182, 61–76 (2008).

  13. 13.

    , , , & Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat. Cell Biol. 11, 228–234 (2009).

  14. 14.

    et al. Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev. 20, 2202–2207 (2006).

  15. 15.

    et al. DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs. Nat. Cell Biol. 9, 604–611 (2007).

  16. 16.

    et al. Maturation of microRNA is hormonally regulated by a nuclear receptor. Mol. Cell 36, 340–347 (2009).

  17. 17.

    , , & SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454, 56–61 (2008).

  18. 18.

    , , & Post-transcriptional regulation of microRNA expression. RNA 12, 1161–1167 (2006).

  19. 19.

    & The let-7 family of microRNAs. Trends Cell Biol. 18, 505–516 (2008).

  20. 20.

    , & Selective blockade of microRNA processing by Lin28. Science 320, 97–100 (2008).

  21. 21.

    , & Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA 14, 1539–1549 (2008).

  22. 22.

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

  23. 23.

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

  24. 24.

    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).

  25. 25.

    , & Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).

  26. 26.

    & Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat. Rev. Mol. Cell Biol. 10, 741–754 (2009).

  27. 27.

    & The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat. Struct. Mol. Biol. 14, 591–596 (2007).

  28. 28.

    , , & Posttranscriptional regulation of miRNAs harboring conserved terminal loops. Mol. Cell 32, 383–393 (2008).

  29. 29.

    & RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 13, 1197–1204 (1994).

  30. 30.

    , , & A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes Dev. 11, 1023–1036 (1997).

  31. 31.

    et al. LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages. FASEB J. 23, 2898–2908 (2009).

  32. 32.

    et al. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 459, 1010–1014 (2009).

  33. 33.

    , , & hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62, 289–321 (1993).

  34. 34.

    , , , & Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 Å resolution. Nat. Struct. Biol. 4, 215–222 (1997).

  35. 35.

    , , , & Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs. Structure 5, 559–570 (1997).

  36. 36.

    & Cooperative-binding and splicing-repressive properties of hnRNP A1. Mol. Cell. Biol. 29, 5620–5631 (2009).

  37. 37.

    , & Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025 (2009).

  38. 38.

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

  39. 39.

    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).

  40. 40.

    , , , & RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res. 31, 5469–5482 (2003).

  41. 41.

    & Molecular architecture of CAG repeats in human disease related transcripts. J. Mol. Biol. 340, 665–679 (2004).

  42. 42.

    & Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry 29, 10717–10722 (1990).

  43. 43.

    & Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc. Natl. Acad. Sci. USA 87, 8403–8407 (1990).

  44. 44.

    & Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc. Natl. Acad. Sci. USA 89, 895–899 (1992).

  45. 45.

    et al. Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling. BMC Mol. Biol. 8, 28 (2007).

  46. 46.

    et al. Modulation of microRNA processing by p53. Nature 460, 529–533 (2009).

  47. 47.

    , & Regulating the regulators: mechanisms controlling the maturation of microRNAs. Trends Biotechnol. 27, 27–36 (2009).

  48. 48.

    et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).

  49. 49.

    et al. Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins. J. Mol. Biol. 332, 85–98 (2003).

  50. 50.

    et al. The NF90–NF45 complex functions as a negative regulator in the microRNA processing pathway. Mol. Cell. Biol. 29, 3754–3769 (2009).

  51. 51.

    et al. A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol. Cell. Proteomics 7, 2019–2027 (2008).

  52. 52.

    et al. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc. Natl. Acad. Sci. USA 105, 3903–3908 (2008).

  53. 53.

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

  54. 54.

    et al. Prospective detection of preclinical lung cancer: results from two studies of heterogeneous nuclear ribonucleoprotein A2/B1 overexpression. Clin. Cancer Res. 3, 2237–2246 (1997).

  55. 55.

    , , , & Altered expression of heterogenous nuclear ribonucleoproteins and SR factors in human colon adenocarcinomas. Cancer Res. 58, 5818–5824 (1998).

  56. 56.

    et al. hnRNP proteins and splicing control. Adv. Exp. Med. Biol. 623, 123–147 (2007).

  57. 57.

    , & A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells. Protein Expr. Purif. 42, 54–58 (2005).

  58. 58.

    , , , & Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J. Cell Biol. 138, 225–238 (1997).

Download references

Acknowledgements

We are grateful to N. Hastie and S. Macias for comments and critical reading of the manuscript, H. Kooshapur (Technical University Munich) and M. Sattler (Technical University Munich) for the generous gift of recombinant UP1 protein and D. Black (Univ. of California, Los Angeles) for providing a KSRP expression vector. This work was supported by Core funding from the Medical Research Council and a project grant from the Wellcome Trust, with additional funds from Eurasnet (European Alternative splicing Network-FP6).

Author information

Affiliations

  1. Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK.

    • Gracjan Michlewski
    •  & Javier F Cáceres

Authors

  1. Search for Gracjan Michlewski in:

  2. Search for Javier F Cáceres in:

Contributions

G.M. and J.F.C. conceived, designed and interpreted the experiments; G.M. performed the experiments and data analysis; J.F.C. supervised the whole project; both authors co-wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Javier F Cáceres.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Methods

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.1874

Further reading