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.

Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction

Nature Structural & Molecular Biology volume 18, pages 796–804 (2011)Cite this article

Subjects

Abstract

We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro–selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R10GGGGR15 at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity–determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G•C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the recognition of other genomic G-rich RNAs.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Sequence and NMR spectra of sc1 RNA and RGG peptide.
Figure 2: RNA resonance assignments in the RGG peptide–sc1 RNA complex.
Figure 3: Identification of G-tetrad alignments and assignment of through-bond correlations in the RGG peptide–sc1 RNA complex.
Figure 4: Solution structure of the RGG peptide–sc1 RNA complex and the architecture of the G-quadruplex and duplex-quadruplex junction.
Figure 5: Architecture of the G-quadruplex and duplex-quadruplex junction.
Figure 6: Details of intermolecular peptide-RNA interactions in the solution structure of the RGG peptide–sc1 RNA complex.
Figure 7: Assessment of the molecular determinants of the peptide and of the RNA for the FMRP RGG peptide–sc1 RNA interaction by filter-binding assay.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Huppert, J.L. & Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908–2916 (2005).

    Article  CAS  Google Scholar 

  2. Todd, A.K., Johnston, M. & Neidle, S. Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res. 33, 2901–2907 (2005).

    Article  CAS  Google Scholar 

  3. Kumari, S., Bugaut, A., Huppert, J.L. & Balasubramanian, S. An RNA G-quadruplex in the 5′ UTR of the NRAS proto-oncogene modulates translation. Nat. Chem. Biol. 3, 218–221 (2007).

    Article  CAS  Google Scholar 

  4. Saxena, S., Miyoshi, D. & Sugimoto, N. Sole and stable RNA duplexes of G-rich sequences located in the 5′-untranslated region of protooncogenes. Biochemistry 49, 7190–7201 (2010).

    Article  CAS  Google Scholar 

  5. Bonnal, S. et al. A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons. J. Biol. Chem. 278, 39330–39336 (2003).

    Article  CAS  Google Scholar 

  6. Oliver, A.W., Bogdarina, I., Schroeder, E., Taylor, I.A. & Kneale, G.G. Preferential binding of fd gene 5 protein to tetraplex nucleic acid structures. J. Mol. Biol. 301, 575–584 (2000).

    Article  CAS  Google Scholar 

  7. Arora, A. et al. Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif. RNA 14, 1290–1296 (2008).

    Article  CAS  Google Scholar 

  8. Bensaid, M. et al. FRAXE-associated mental retardation protein (FMR2) is an RNA-binding protein with high affinity for G-quartet RNA forming structure. Nucleic Acids Res. 37, 1269–1279 (2009).

    Article  CAS  Google Scholar 

  9. Didiot, M.C. et al. The G-quartet containing FMRP binding site in FMR1 mRNA is a potent exonic splicing enhancer. Nucleic Acids Res. 36, 4902–4912 (2008).

    Article  CAS  Google Scholar 

  10. 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 

  11. Bagga, P.S., Ford, L.P., Chen, F. & Wilusz, J. The G-rich auxiliary downstream element has distinct sequence and position requirements and mediates efficient 3′ end pre-mRNA processing through a trans-acting factor. Nucleic Acids Res. 23, 1625–1631 (1995).

    Article  CAS  Google Scholar 

  12. Darnell, J.C. et al. Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function. Cell 107, 489–499 (2001).

    Article  CAS  Google Scholar 

  13. Schaeffer, C. et al. The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif. EMBO J. 20, 4803–4813 (2001).

    Article  CAS  Google Scholar 

  14. Bassell, G.J. & Warren, S.T. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron 60, 201–214 (2008).

    Article  CAS  Google Scholar 

  15. De Boulle, K. et al. A point mutation in the FMR-1 gene associated with fragile X mental retardation. Nat. Genet. 3, 31–35 (1993).

    Article  CAS  Google Scholar 

  16. Zang, J.B. et al. A mouse model of the human Fragile X syndrome I304N mutation. PLoS Genet. 5, e1000758 (2009).

    Article  Google Scholar 

  17. Darnell, J.C., Fraser, C.E., Mostovetsky, O. & Darnell, R.B. Discrimination of common and unique RNA-binding activities among Fragile X mental retardation protein paralogs. Hum. Mol. Genet. 18, 3164–3177 (2009).

    Article  CAS  Google Scholar 

  18. Phan, A.T. & Patel, D.J. A site-specific low-enrichment (15)N,(13)C isotope-labeling approach to unambiguous NMR spectral assignments in nucleic acids. J. Am. Chem. Soc. 124, 1160–1161 (2002).

    Article  CAS  Google Scholar 

  19. Phan, A.T. Long-range imino proton-13C J-couplings and the through-bond correlation of imino and non-exchangeable protons in unlabeled DNA. J. Biomol. NMR 16, 175–178 (2000).

    Article  CAS  Google Scholar 

  20. Martadinata, H. & Phan, A.T. Structure of propeller-type parallel-stranded RNA G-quadruplexes, formed by human telomeric RNA sequences in K+ solution. J. Am. Chem. Soc. 131, 2570–2578 (2009).

    Article  CAS  Google Scholar 

  21. Fiala, R., Jiang, F. & Sklenar, V. Sensitivity optimized HCN and HCNCH experiments for 13C/15N labeled oligonucleotides. J. Biomol. NMR 12, 373–383 (1998).

    Article  CAS  Google Scholar 

  22. Nikonowicz, E.P. & Pardi, A. An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids. J. Mol. Biol. 232, 1141–1156 (1993).

    Article  CAS  Google Scholar 

  23. Dingley, J.C. & Grzesiek, S. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J. Am. Chem. Soc. 120, 8293–8297 (1998).

    Article  CAS  Google Scholar 

  24. Pervushin, K. et al. NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy. Proc. Natl. Acad. Sci. USA 95, 14147–14151 (1998).

    Article  CAS  Google Scholar 

  25. Majumdar, A., Kettani, A., Skripkin, E. & Patel, D. Observation of internucleotide NH.N hydrogen bonds in the absence of directly detectable protons. J. Biomol. NMR 15, 207–211 (1999).

    Article  CAS  Google Scholar 

  26. Majumdar, A., Kettani, A. & Skripkin, E. Observation and measurement of internucleotide 2JNN coupling constants between 15N nuclei with widely separated chemical shifts. J. Biomol. NMR 14, 67–70 (1999).

    Article  CAS  Google Scholar 

  27. Muhandiram, R. & Kay, L.E. Gradient-enhanced triple-resonance three-dimensional NMR experiments with improved sensitivity. J. Magn. Reson. B. 103, 203–216 (1994).

    Article  CAS  Google Scholar 

  28. Clore, G.M. & Gronenborn, A.M. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239, 349–363 (1994).

    Article  CAS  Google Scholar 

  29. Lunde, B.M., Moore, C. & Varani, G. RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell Biol. 8, 479–490 (2007).

    Article  CAS  Google Scholar 

  30. Majumdar, A., Gosser, Y. & Patel, D.J. 1H–1H correlations across N-H...N hydrogen bonds in nucleic acids. J. Biomol. NMR 21, 289–306 (2001).

    Article  CAS  Google Scholar 

  31. Parkinson, G.N., Lee, M.P. & Neidle, S. Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417, 876–880 (2002).

    Article  CAS  Google Scholar 

  32. Phan, A.T., Modi, Y.S. & Patel, D.J. Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter. J. Am. Chem. Soc. 126, 8710–8716 (2004).

    Article  CAS  Google Scholar 

  33. 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 

  34. Ramos, A., Hollingworth, D. & Pastore, A. G-quartet-dependent recognition between the FMRP RGG box and RNA. RNA 9, 1198–1207 (2003).

    Article  CAS  Google Scholar 

  35. Zanotti, K.J., Lackey, P.E., Evans, G.L. & Mihailescu, M.R. Thermodynamics of the fragile X mental retardation protein RGG box interactions with G quartet forming RNA. Biochemistry 45, 8319–8330 (2006).

    Article  CAS  Google Scholar 

  36. Blackwell, E., Zhang, X. & Ceman, S. Arginines of the RGG box regulate FMRP association with polyribosomes and mRNA. Hum. Mol. Genet. 19, 1314–1323 (2010).

    Article  CAS  Google Scholar 

  37. Cate, J.H. et al. RNA tertiary structure mediation by adenosine platforms. Science 273, 1696–1699 (1996).

    Article  CAS  Google Scholar 

  38. Puglisi, J.D., Chen, L., Blanchard, S. & Frankel, A.D. Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex. Science 270, 1200–1203 (1995).

    Article  CAS  Google Scholar 

  39. Ye, X., Kumar, R.A. & Patel, D.J. Molecular recognition in the bovine immunodeficiency virus Tat peptide-TAR RNA complex. Chem. Biol. 2, 827–840 (1995).

    Article  CAS  Google Scholar 

  40. Davidson, A. et al. Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein. Proc. Natl. Acad. Sci. USA 106, 11931–11936 (2009).

    Article  CAS  Google Scholar 

  41. Licatalosi, D.D. & Darnell, R.B. Splicing regulation in neurologic disease. Neuron 52, 93–101 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Ota, T. et al. Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40–45 (2004).

    Article  Google Scholar 

  44. Vaccari, T. et al. The human gene coding for HCN2, a pacemaker channel of the heart. Biochim. Biophys. Acta 1446, 419–425 (1999).

    Article  CAS  Google Scholar 

  45. Wang, C. & Meier, U.T. Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins. EMBO J. 23, 1857–1867 (2004).

    Article  CAS  Google Scholar 

  46. Vanhalst, K., Kools, P., Staes, K., van Roy, F. & Redies, C. delta-Protocadherins: a gene family expressed differentially in the mouse brain. Cell. Mol. Life Sci. 62, 1247–1259 (2005).

    Article  CAS  Google Scholar 

  47. Gerdes, D., Wehling, M., Leube, B. & Falkenstein, E. Cloning and tissue expression of two putative steroid membrane receptors. Biol. Chem. 379, 907–911 (1998).

    CAS  PubMed  Google Scholar 

  48. Katoh, K. et al. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J. Biol. Chem. 278, 39104–39113 (2003).

    Article  CAS  Google Scholar 

  49. Hanakahi, L.A., Sun, H. & Maizels, N. High affinity interactions of nucleolin with G-G-paired rDNA. J. Biol. Chem. 274, 15908–15912 (1999).

    Article  CAS  Google Scholar 

  50. Mazroui, R. et al. Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression. Hum. Mol. Genet. 11, 3007–3017 (2002).

    Article  CAS  Google Scholar 

  51. Stetler, A. et al. Identification and characterization of the methyl arginines in the fragile X mental retardation protein Fmrp. Hum. Mol. Genet. 15, 87–96 (2006).

    Article  CAS  Google Scholar 

  52. Pfeiffer, B.E. et al. Fragile X mental retardation protein is required for synapse elimination by the activity-dependent transcription factor MEF2. Neuron 66, 191–197 (2010).

    Article  CAS  Google Scholar 

  53. Batey, R.T., Battiste, J.L. & Williamson, J.R. Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol. 261, 300–322 (1995).

    Article  CAS  Google Scholar 

  54. Pikovskaya, O., Serganov, A.A., Polonskaia, A., Serganov, A. & Patel, D.J. Preparation and crystallization of riboswitch-ligand complexes. Methods Mol. Biol. 540, 115–128 (2009).

    Article  CAS  Google Scholar 

  55. Kuryavyi, V. & Patel, D.J. Solution structure of a unique G-quadruplex scaffold adopted by a guanosine-rich human intronic sequence. Structure 18, 73–82 (2010).

    Article  CAS  Google Scholar 

  56. Brown, V. et al. Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107, 477–487 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Gosser and S. Pitt (Memorial Sloan-Kettering Cancer Center) for their participation at the early stage of the project, O. Mostovetsky, E.F. Stone and K.Y.S. Hung (Rockefeller University) for technical assistance, and A. Lash (Memorial Sloan-Kettering Cancer Center) for bioinformatics of the RG4R motif in the human genome. This research was supported by US National Institutes of Health grants CA049982 (to D.J.P.) and R01 HD040647 (to J.C.D.) and Singapore Biomedical Research Council grant 07/1/22/19/542 to A.T.P. D.J.P. is a member of the New York Structural Biology Center, supported by US National Institutes of Health grant GM66354.

Author information

Author notes

  1. Anh Tuân Phan, Vitaly Kuryavyi and Jennifer C Darnell: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Anh Tuân Phan, Vitaly Kuryavyi, Alexander Serganov, Serge Ilin, Tanya Raslin, Anna Polonskaia & Dinshaw J Patel

  2. School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore

    Anh Tuân Phan

  3. Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA

    Jennifer C Darnell, Cynthia Chen, David Clain & Robert B Darnell

  4. Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA

    Robert B Darnell

  5. Biomolecular NMR Center, Johns Hopkins University, Baltimore, Maryland, USA

    Ananya Majumdar

Authors
  1. Anh Tuân Phan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vitaly Kuryavyi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer C Darnell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander Serganov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ananya Majumdar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Serge Ilin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tanya Raslin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Anna Polonskaia
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Cynthia Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. David Clain
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Robert B Darnell
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dinshaw J Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.T.P., A.M. and S.I. were responsible for NMR studies, V.K. undertook the computations, and A.S., T.R. and A.P. prepared labeled NMR samples, all under the supervision of D.J.P. C.C., D.C. and J.C.D. did the filter-binding assays under the joint supervision of J.C.D. and R.B.D. The paper was written jointly by D.J.P., A.T.P., V.K., J.C.D. and R.B.D.

Corresponding authors

Correspondence to Anh Tuân Phan, Jennifer C Darnell or Dinshaw J Patel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary Discussion and Supplementary Methods (PDF 9510 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Phan, A., Kuryavyi, V., Darnell, J. et al. Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction. Nat Struct Mol Biol 18, 796–804 (2011). https://doi.org/10.1038/nsmb.2064

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2064

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