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 G-tract recognition and encaging by hnRNP F quasi-RRMs

Abstract

The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. We have determined the solution structures of the three quasi–RNA-recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, with the G-tract 'encaged', making the qRRM a novel RNA binding domain. We defined a consensus signature sequence for qRRMs and identified other human qRRM-containing proteins that also specifically recognize G-tract RNAs. Our structures explain how qRRMs can sequester G-tracts, maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the Bcl-x pre-mRNA, suggesting that hnRNP F would act by remodeling RNA secondary and tertiary structures.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Overview of the structures and ITC of human hnRNP F qRRM1–AGGGAU, qRRM2–AGGGAU and qRRM3–AGGGAU.
Figure 2: Specific recognition of G-tract RNA by hnRNP F qRRMs.
Figure 3: A consensus qRRM motif is present in human proteins that are not hnRNP F family members.
Figure 4: hnRNP F qRRMs prevent RNA structure formation.
Figure 5: Activity of the qRRMs of hnRNP F on splice-site selection.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

GenBank/EMBL/DDBJ

References

  1. Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

  2. Cooper, T.A., Wan, L. & Dreyfuss, G. RNA and disease. Cell 136, 777–793 (2009).

    Article  CAS  Google Scholar 

  3. Wang, Z. et al. Systematic identification and analysis of exonic splicing silencers. Cell 119, 831–845 (2004).

    Article  CAS  Google Scholar 

  4. Yeo, G., Hoon, S., Venkatesh, B. & Burge, C.B. Variation in sequence and organization of splicing regulatory elements in vertebrate genes. Proc. Natl. Acad. Sci. USA 101, 15700–15705 (2004).

    Article  CAS  Google Scholar 

  5. Zarudnaya, M.I., Kolomiets, I.M., Potyahaylo, A.L. & Hovorun, D.M. Downstream elements of mammalian pre-mRNA polyadenylation signals: primary, secondary and higher-order structures. Nucleic Acids Res. 31, 1375–1386 (2003).

    Article  CAS  Google Scholar 

  6. Huppert, J.L., Bugaut, A., Kumari, S. & Balasubramanian, S. G-quadruplexes: the beginning and end of UTRs. Nucleic Acids Res. 36, 6260–6268 (2008).

    Article  CAS  Google Scholar 

  7. Xiao, X. et al. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat. Struct. Mol. Biol. 16, 1094–1100 (2009).

    Article  CAS  Google Scholar 

  8. Caputi, M. & Zahler, A.M. Determination of the RNA binding specificity of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/H′/F/2H9 family. J. Biol. Chem. 276, 43850–43859 (2001).

    Article  CAS  Google Scholar 

  9. Matunis, M.J., Xing, J. & Dreyfuss, G. The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization. Nucleic Acids Res. 22, 1059–1067 (1994).

    Article  CAS  Google Scholar 

  10. Park, Y.W., Wilusz, J. & Katze, M.G. Regulation of eukaryotic protein synthesis: selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1. Proc. Natl. Acad. Sci. USA 96, 6694–6699 (1999).

    Article  CAS  Google Scholar 

  11. Ufer, C. et al. Translational regulation of glutathione peroxidase 4 expression through guanine-rich sequence-binding factor 1 is essential for embryonic brain development. Genes Dev. 22, 1838–1850 (2008).

    Article  CAS  Google Scholar 

  12. Cobbold, L.C. et al. Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs. Mol. Cell. Biol. 28, 40–49 (2008).

    Article  CAS  Google Scholar 

  13. Min, H., Chan, R.C. & Black, D.L. The generally expressed hnrRNP F is involved in a neural-specific pre-mRNA splicing event. Genes Dev. 9, 2659–2671 (1995).

    Article  CAS  Google Scholar 

  14. Chen, C.D., Kobayashi, R. & Helfman, D.M. Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat β-tropomyosin gene. Genes Dev. 13, 593–606 (1999).

    Article  CAS  Google Scholar 

  15. Jacquenet, S. et al. A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H. J. Biol. Chem. 276, 40464–40475 (2001).

    Article  CAS  Google Scholar 

  16. Caputi, M. & Zahler, A.M. SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D. EMBO J. 21, 845–855 (2002).

    Article  CAS  Google Scholar 

  17. Garneau, D., Revil, T., Fisette, J.F. & Chabot, B. hnRNP F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x. J. Biol. Chem. 280, 22641–22650 (2005).

    Article  CAS  Google Scholar 

  18. Camats, M., Guil, S., Kokolo, M. & Bach-Elias, M. P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras. PLoS One 3, e2926 (2008).

    Article  Google Scholar 

  19. Coles, J.L., Hallegger, M. & Smith, C.W.J. A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F. RNA 15, 33–43 (2009).

    Article  CAS  Google Scholar 

  20. Hai, Y. et al. A G-tract element in apoptotic agents-induced alternative splicing. Nucleic Acids Res. 36, 3320–3331 (2008).

    Article  CAS  Google Scholar 

  21. Martinez-Contreras, R. et al. Intronic binding sites for hnRNP A/B and hnRNP F/H proteins stimulate pre-mRNA splicing. PLoS Biol. 4, e21 (2006).

    Article  Google Scholar 

  22. Mauger, D.M., Lin, C. & Garcia-Blanco, M.A. HnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc. Mol. Cell. Biol. 28, 5403–5419 (2008).

    Article  CAS  Google Scholar 

  23. Oberg, D., Fay, J., Lambkin, H. & Schwartz, S. A downstream polyadenylation element in human papillomavirus type 16 L2 encodes multiple GGG motifs and interacts with hnRNP H. J. Virol. 79, 9254–9269 (2005).

    Article  Google Scholar 

  24. Qian, Z.W. & Wilusz, J. An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal. Mol. Cell. Biol. 11, 5312–5320 (1991).

    Article  CAS  Google Scholar 

  25. Buratti, E. et al. hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes. Nucleic Acids Res. 32, 4224–4236 (2004).

    Article  CAS  Google Scholar 

  26. Cogan, J.D. et al. A novel mechanism of aberrant pre-mRNA splicing in humans. Hum. Mol. Genet. 6, 909–912 (1997).

    Article  CAS  Google Scholar 

  27. Lew, J.M. et al. CDKN1C mutation in Wiedemann-Beckwith syndrome patients reduces RNA splicing efficiency and identifies a splicing enhancer. Am. J. Med. Genet. A. 127A, 268–276 (2004).

    Article  Google Scholar 

  28. Masuda, A. et al. hnRNP H enhances skipping of a nonfunctional exon P3A in CHRNA1 and a mutation disrupting its binding causes congenital myasthenic syndrome. Hum. Mol. Genet. 17, 4022–4035 (2008).

    Article  CAS  Google Scholar 

  29. Pagani, F., Buratti, E., Stuani, C. & Baralle, F.E. Missense, nonsense, and neutral mutations define juxtaposed regulatory elements of splicing in cystic fibrosis transmembrane regulator exon 9. J. Biol. Chem. 278, 26580–26588 (2003).

    Article  CAS  Google Scholar 

  30. Van Laer, L. et al. Nonsyndromic hearing impairment is associated with a mutation in DFNA5. Nat. Genet. 20, 194–197 (1998).

    Article  CAS  Google Scholar 

  31. Barberan-Soler, S. & Zahler, A.M. Alternative splicing regulation during C. elegans development: splicing factors as regulated targets. PLoS Genet. 4, e1000001 (2008).

    Article  Google Scholar 

  32. Chang, L.Y., Ali, A.R., Hassan, S.S. & AbuBakar, S. Human neuronal cell protein responses to Nipah virus infection. Virol. J. 4, 54 (2007).

    Article  Google Scholar 

  33. Honoré, B., Baandrup, U. & Vorum, H. Heterogeneous nuclear ribonucleoproteins F and H/H′ show differential expression in normal and selected cancer tissues. Exp. Cell Res. 294, 199–209 (2004).

    Article  Google Scholar 

  34. Boise, L.H. et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74, 597–608 (1993).

    Article  CAS  Google Scholar 

  35. Han, K., Yeo, G., An, P., Burge, C.B. & Grabowski, P.J. A combinatorial code for splicing silencing: UAGG and GGGG motifs. PLoS Biol. 3, e158 (2005).

    Article  Google Scholar 

  36. Crawford, J.B. & Patton, J.G. Activation of α-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F. Mol. Cell. Biol. 26, 8791–8802 (2006).

    Article  CAS  Google Scholar 

  37. Schaub, M.C., Lopez, S.R. & Caputi, M. Members of the heterogeneous nuclear ribonucleoprotein H family activate splicing of an HIV-1 splicing substrate by promoting formation of ATP-dependent spliceosomal complexes. J. Biol. Chem. 282, 13617–13626 (2007).

    Article  CAS  Google Scholar 

  38. Jablonski, J.A., Buratti, E., Stuani, C. & Caputi, M. The secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity. J. Virol. 82, 8038–8050 (2008).

    Article  CAS  Google Scholar 

  39. Honoré, B. et al. Heterogeneous nuclear ribonucleoproteins H, H′, and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes. J. Biol. Chem. 270, 28780–28789 (1995).

    Article  Google Scholar 

  40. Dominguez, C. & Allain, F.H. NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition. Nucleic Acids Res. 34, 3634–3645 (2006).

    Article  CAS  Google Scholar 

  41. Wenter, P., Reymond, L., Auweter, S.D., Allain, F.H. & Pitsch, S. Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes. Nucleic Acids Res. 34, e79 (2006).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Clery, A., Blatter, M. & Allain, F.H.T. RNA recognition motifs: boring? Not quite. Curr. Opin. Struct. Biol. 18, 290–298 (2008).

    Article  CAS  Google Scholar 

  44. Ding, J. et al. Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. Genes Dev. 13, 1102–1115 (1999).

    Article  CAS  Google Scholar 

  45. Warzecha, C.C., Sato, T.K., Nabet, B., Hogenesch, J.B. & Carstens, R.P. ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol. Cell 33, 591–601 (2009).

    Article  CAS  Google Scholar 

  46. Jin, S.B. et al. Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal subunits in yeast. J. Biol. Chem. 277, 18431–18439 (2002).

    Article  CAS  Google Scholar 

  47. Enokizono, Y. et al. Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D. J. Biol. Chem. 280, 18862–18870 (2005).

    Article  CAS  Google Scholar 

  48. Hiller, M., Zhang, Z., Backofen, R. & Stamm, S. Pre-mRNA secondary structures influence exon recognition. PLoS Genet. 3, e204 (2007).

    Article  Google Scholar 

  49. Warf, M.B. & Berglund, J.A. Role of RNA structure in regulating pre-mRNA splicing. Trends Biochem. Sci. 35, 169–178 (2010).

    Article  CAS  Google Scholar 

  50. Kikin, O., D'Antonio, L. & Bagga, P.S. QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res. 34, W676–W682 (2006).

    Article  CAS  Google Scholar 

  51. Patel, D.J., Phan, A.T. & Kuryavyi, V. Human telomere, oncogenic promoter and 5′-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics. Nucleic Acids Res. 35, 7429–7455 (2007).

    Article  CAS  Google Scholar 

  52. Roy, D. & Lieber, M.R. G clustering is important for the initiation of transcription-induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol. Cell. Biol. 29, 3124–3133 (2009).

    Article  CAS  Google Scholar 

  53. Aguilera, A. & Gomez-Gonzalez, B. Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet. 9, 204–217 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  55. 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  Google Scholar 

  56. Herrmann, T., Guntert, P. & Wuthrich, K. Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS. J. Biomol. NMR 24, 171–189 (2002).

    Article  CAS  Google Scholar 

  57. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  59. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

    Article  CAS  Google Scholar 

  60. Chabot, B., Blanchette, M., Lapierre, I. & La Branche, H. An intron element modulating 5′ splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1. Mol. Cell. Biol. 17, 1776–1786 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank D. Black (Howard Hugues Medical Institute) for providing the clone of full-length hnRNP F, S. Pitsch (Ecole Polytechnique Fédérale de Lausanne) for providing sugar-13C-labeled RNAs, F. Oberstrass for his implication in the optimization of the conditions for complex formation, J. Boudet for his help with ITC measurements, M. Crespo and R. Glockshuber for their help with CD measurements, C. Maris for his help with UV measurements, L. Shkreta for testing qRRM2 of RBM19 in splicing, S. Jayne and D. Theler for critical reading of the manuscript and members of the group for helpful discussions. C.D. was supported by postdoctoral fellowships from the Roche Research Foundation for Biology and from the Novartis Research Foundation. Financial support from the Swiss National Science Foundation, the Structural Biology National Center of Competence in Research and the European Alternative Splicing Network is also acknowledged.

Author information

Authors and Affiliations

Authors

Contributions

F.H.-T.A. and B.C. designed the project; C.D. prepared protein and RNA samples for structural studies; C.D. and F.H.-T.A. analyzed NMR data; C.D. performed structure calculations and UV measurements; J.F.F. performed in vitro splicing assays; C.D., J.F.F., B.C. and F.H.-T.A. wrote the manuscript; all authors discussed the results and approved the manuscript.

Corresponding author

Correspondence to 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 (PDF 16165 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dominguez, C., Fisette, JF., Chabot, B. et al. Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. Nat Struct Mol Biol 17, 853–861 (2010). https://doi.org/10.1038/nsmb.1814

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1814

This article is cited by

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