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.

An RNA map predicting Nova-dependent splicing regulation

Abstract

Nova proteins are neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova’s effect to inhibit or enhance exon inclusion, which led us to examine the relationship between the map and Nova’s mechanism of action. Nova binding to an exonic YCAY cluster changed the protein complexes assembled on pre-mRNA, blocking U1 snRNP (small nuclear ribonucleoprotein) binding and exon inclusion, whereas Nova binding to an intronic YCAY cluster enhanced spliceosome assembly and exon inclusion. Assays of splicing intermediates of Nova-regulated transcripts in mouse brain revealed that Nova preferentially regulates removal of introns harbouring (or closest to) YCAY clusters. These results define a genome-wide map relating the position of a cis-acting element to its regulation by an RNA binding protein, namely that Nova binding to YCAY clusters results in a local and asymmetric action to regulate spliceosome assembly and alternative splicing in neurons.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Definition of the Nova–RNA binding map.
Figure 2: YCAY cluster position predicts Nova-dependent splicing regulation.
Figure 3: Nova inhibits splicing of an RNA substrate containing a NESS2 element.
Figure 4: Nova binding to a splicing enhancer promotes spliceosome assembly.
Figure 5: Analysis of splicing intermediates in Nova-regulated RNAs.
Figure 6: An RNA map providing a comprehensive view of Nova function.

References

  1. Hallikas, O. et al. Genome-wide prediction of mammalian enhancers based on analysis of transcription-factor binding affinity. Cell 124, 47–59 (2006)

    CAS  PubMed  Google Scholar 

  2. Wasserman, W. W. & Sandelin, A. Applied bioinformatics for the identification of regulatory elements. Nature Rev. Genet. 5, 276–287 (2004)

    CAS  PubMed  Google Scholar 

  3. Fairbrother, W. G., Yeh, R. F., Sharp, P. A. & Burge, C. B. Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007–1013 (2002)

    ADS  CAS  PubMed  Google Scholar 

  4. Hui, J. et al. Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing. EMBO J. 24, 1988–1998 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Liu, H. X., Zhang, M. & Krainer, A. R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 12, 1998–2012 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  7. Yeo, G. W., Van Nostrand, E., Holste, D., Poggio, T. & Burge, C. B. Identification and analysis of alternative splicing events conserved in human and mouse. Proc. Natl Acad. Sci. USA 102, 2850–2855 (2005)

    ADS  CAS  PubMed  Google Scholar 

  8. Goren, A. et al. Comparative analysis identifies exonic splicing regulatory sequences—The complex definition of enhancers and silencers. Mol. Cell 22, 769–781 (2006)

    CAS  PubMed  Google Scholar 

  9. Ryder, S. P., Frater, L. A., Abramovitz, D. L., Goodwin, E. B. & Williamson, J. R. RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1. Nature Struct. Mol. Biol. 11, 20–28 (2004)

    CAS  Google Scholar 

  10. 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)

    PubMed  PubMed Central  Google Scholar 

  11. Ule, J. & Darnell, R. B. RNA binding proteins and the regulation of neuronal synaptic plasticity. Curr. Opin. Neurobiol. 16, 102–110 (2006)

    CAS  PubMed  Google Scholar 

  12. Licatalosi, D. D. & Darnell, R. B. Splicing regulation in neurologic disease. Neuron 52, (1)93–101 (2006)

    CAS  PubMed  Google Scholar 

  13. Buckanovich, R. J., Yang, Y. Y. & Darnell, R. B. The onconeural antigen Nova-1 is a neuron-specific RNA-binding protein, the activity of which is inhibited by paraneoplastic antibodies. J. Neurosci. 16, 1114–1122 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Jensen, K. B. et al. Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron 25, 359–371 (2000)

    CAS  PubMed  Google Scholar 

  15. Yang, Y. Y., Yin, G. L. & Darnell, R. B. The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia. Proc. Natl Acad. Sci. USA 95, 13254–13259 (1998)

    ADS  CAS  PubMed  Google Scholar 

  16. Buckanovich, R. J. & Darnell, R. B. The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.. Mol. Cell. Biol. 17, 3194–3201 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Jensen, K. B., Musunuru, K., Lewis, H. A., Burley, S. K. & Darnell, R. B. The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain. Proc. Natl Acad. Sci. USA 97, 5740–5745 (2000)

    ADS  CAS  PubMed  Google Scholar 

  18. Lewis, H. A. et al. Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome. Cell 100, 323–332 (2000)

    CAS  PubMed  Google Scholar 

  19. Dredge, B. K. & Darnell, R. B. Nova regulates GABAA receptor γ2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer. Mol. Cell. Biol. 23, 4687–4700 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Dredge, B. K., Stefani, G., Engelhard, C. C. & Darnell, R. B. Nova autoregulation reveals dual functions in neuronal splicing. EMBO J. 24, 1608–1620 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Ule, J. et al. CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 (2003)

    ADS  CAS  PubMed  Google Scholar 

  22. Ule, J. et al. Nova regulates brain-specific splicing to shape the synapse. Nature Genet. 37, 844–852 (2005)

    CAS  PubMed  Google Scholar 

  23. Michaud, S. & Reed, R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 5, 2534–2546 (1991)

    CAS  PubMed  Google Scholar 

  24. Bennett, M., Michaud, S., Kingston, J. & Reed, R. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 6, 1986–2000 (1992)

    CAS  PubMed  Google Scholar 

  25. Sharma, S., Falick, A. M. & Black, D. L. Polypyrimidine tract binding protein blocks the 5′ splice site-dependent assembly of U2AF and the prespliceosomal E complex. Mol. Cell 19, 485–496 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Tarn, W. Y. & Steitz, J. A. proteins can compensate for the loss of U1 snRNP functions in vitro.. Genes Dev. 8, 2704–2717 (1994)

    CAS  PubMed  Google Scholar 

  27. Barabino, S. M., Blencowe, B. J., Ryder, U., Sproat, B. S. & Lamond, A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell 63, 293–302 (1990)

    CAS  PubMed  Google Scholar 

  28. Query, C. C., McCaw, P. S. & Sharp, P. A. A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract. Mol. Cell. Biol. 17, 2944–2953 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Buratti, E. & Baralle, F. E. Influence of RNA secondary structure on the pre-mRNA splicing process. Mol. Cell. Biol. 24, 10505–10514 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Xing, Y. & Lee, C. Alternative splicing and RNA selection pressure—evolutionary consequences for eukaryotic genomes. Nature Rev. Genet. 7, 499–509 (2006)

    CAS  PubMed  Google Scholar 

  31. Coulter, L. R., Landree, M. A. & Cooper, T. A. Identification of a new class of exonic splicing enhancers by in vivo selection. Mol. Cell. Biol. 17, 2143–2150 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Gersappe, A. & Pintel, D. J. CA- and purine-rich elements form a novel bipartite exon enhancer which governs inclusion of the minute virus of mice NS2-specific exon in both singly and doubly spliced mRNAs. Mol. Cell. Biol. 19, 364–375 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Polydorides, A. D., Okano, H. J., Yang, Y. Y., Stefani, G. & Darnell, R. B. A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. Proc. Natl Acad. Sci. USA 97, 6350–6355 (2000)

    ADS  CAS  PubMed  Google Scholar 

  34. Valcarcel, J., Singh, R., Zamore, P. D. & Green, M. R. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362, 171–175 (1993)

    ADS  CAS  PubMed  Google Scholar 

  35. Del Gatto-Konczak, F., Olive, M., Gesnel, M. C. & Breathnach, R. hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell. Biol. 19, 251–260 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhu, J., Mayeda, A. & Krainer, A. R. Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. Mol. Cell 8, 1351–1361 (2001)

    CAS  PubMed  Google Scholar 

  37. Izquierdo, J. M. et al. Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. Mol. Cell 19, 475–484 (2005)

    CAS  PubMed  Google Scholar 

  38. Chou, M. Y., Rooke, N., Turck, C. W. & Black, D. L. hnRNP H is a component of a splicing enhancer complex that activates a c-Src alternative exon in neuronal cells. Mol. Cell. Biol. 19, 69–77 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Underwood, J. G., Boutz, P. L., Dougherty, J. D., Stoilov, P. & Black, D. L. Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals. Mol. Cell. Biol. 25, 10005–10016 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Carlo, T., Sterner, D. A. & Berget, S. M. An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon. RNA 2, 342–353 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Lewis, H. A. et al. Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains. Struct. Fold. Des. 7, 191–203 (1999)

    CAS  Google Scholar 

  42. Chou, M. Y., Underwood, J. G., Nikolic, J., Luu, M. H. & Black, D. L. Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-Src neural-specific splicing. Mol. Cell 5, 949–957 (2000)

    CAS  PubMed  Google Scholar 

  43. 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)

    PubMed  PubMed Central  Google Scholar 

  44. Gee, S. L. et al. Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice. Blood 95, 692–699 (2000)

    CAS  PubMed  Google Scholar 

  45. Kessler, O., Jiang, Y. & Chasin, L. A. Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. Mol. Cell. Biol. 13, 6211–6222 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lang, K. M. & Spritz, R. A. In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?. Mol. Cell. Biol. 7, 3428–3437 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Tsai, M. J., Ting, A. C., Nordstrom, J. L., Zimmer, W. & O’Malley, B. W. Processing of high molecular weight ovalbumin and ovomucoid precursor RNAs to messenger RNA. Cell 22, 219–230 (1980)

    CAS  PubMed  Google Scholar 

  48. Cook, H. L. et al. Small nuclear RNAs encoded by Herpesvirus saimiri upregulate the expression of genes linked to T cell activation in virally transformed T cells. Curr. Biol. 15, 974–979 (2005)

    CAS  PubMed  Google Scholar 

  49. Beffert, U. et al. Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2. Neuron 47, 567–579 (2005)

    CAS  PubMed  Google Scholar 

  50. Huang, C. S. et al. Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition. Cell 123, 105–118 (2005)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Eom and D. Licatalosi for help with breeding Nova1–/–Nova2–/– mice and providing mouse brain; P. Ariel for technical assistance; M. Babu, C. Smith, J. Valcarcel, G. Yeo, D. Licatalosi, J. Darnell, S. Xie, and S. W. Chi for critically reading the manuscript; D. Karolchik and J. Jackson for help with the UCSC Genome Bioinformatics tools; J. Okano for the hnRNP K expression construct; A. Krainer and L. Manche for help with in vitro splicing assays; K. Dredge for DNA constructs; D. Black for sharing unpublished results; and M. Konarska and members of the laboratory for discussions. Supported by the NIH (R.B.D.) and the Howard Hughes Medical Institute, the tumour immunology program of Cancer Research Institute (J.U.) and a Human Frontiers Science Program Fellowship (M.R.). R.B.D. is an Investigator of the Howard Hughes Medical Institute. Author Contributions J.U. bioinformatically defined the RNA map, and predicted and analysed Nova-target exons and splicing intermediates; G.S. performed in vitro studies of the mechanisms of Nova action and its effects on spliceosome assembly; A.M. purified PCR products for sequencing; M.R. characterized the Nova1–/–Nova2–/– mice; X.W. wrote the sequence analysis programs; B.T. and T.G. provided the database of alternative exons; B.J.B. provided depleted extracts; and R.B.D. supervised all studies. The manuscript was prepared by J.U., G.S. and R.B.D., with the participation of all authors.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Robert B. Darnell.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–14. (PDF 26155 kb)

Supplementary Figure Legends

This file contains text to accompany the above Supplementary Figures, and additional references. (DOC 203 kb)

Supplementary Methods

This file contains additional details on the methods used in this study. (DOC 436 kb)

Supplementary Tables

This file contains Supplementary Tables 1–3. (PDF 224 kb)

Supplementary Table Legends

This file contains text to accompany the above Supplementary Tables. (DOC 67 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ule, J., Stefani, G., Mele, A. et al. An RNA map predicting Nova-dependent splicing regulation. Nature 444, 580–586 (2006). https://doi.org/10.1038/nature05304

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05304

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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