The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

Article metrics


The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear1. We show here that central nervous system–specific deletion of the gene encoding Rbfox1 results in heightened susceptibility to spontaneous and kainic acid–induced seizures. Electrophysiological recording revealed a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole-transcriptome analyses identified multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Rbfox1−/− brains lack Rbfox1 protein expression but possess normal morphology.
Figure 2: Rbfox1−/− brains are epileptic and hyperexcitable.
Figure 3: Rbfox1−/− brain exhibits splicing changes in transcripts affecting synaptic function and neuronal excitation.


  1. 1

    Kuroyanagi, H. Fox-1 family of RNA-binding proteins. Cell. Mol. Life Sci. 66, 3895–3907 (2009).

  2. 2

    Li, Q., Lee, J.A. & Black, D.L. Neuronal regulation of alternative pre-mRNA splicing. Nat. Rev. Neurosci. 8, 819–831 (2007).

  3. 3

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

  4. 4

    Jin, Y. et al. A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG. EMBO J. 22, 905–912 (2003).

  5. 5

    Nakahata, S. & Kawamoto, S. Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities. Nucleic Acids Res. 33, 2078–2089 (2005).

  6. 6

    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).

  7. 7

    Auweter, S.D. et al. Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO J. 25, 163–173 (2006).

  8. 8

    Black, D.L. Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro. Cell 69, 795–807 (1992).

  9. 9

    Huh, G.S. & Hynes, R.O. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev. 8, 1561–1574 (1994).

  10. 10

    Modafferi, E.F. & Black, D.L. A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. Mol. Cell. Biol. 17, 6537–6545 (1997).

  11. 11

    Zhang, C. et al. Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2. Genes Dev. 22, 2550–2563 (2008).

  12. 12

    Yeo, G.W. et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137 (2009).

  13. 13

    Shibata, H., Huynh, D.P. & Pulst, S.M. A novel protein with RNA-binding motifs interacts with ataxin-2. Hum. Mol. Genet. 9, 1303–1313 (2000).

  14. 14

    Bhalla, K. et al. The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the A2BP1 gene. J. Hum. Genet. 49, 308–311 (2004).

  15. 15

    Barnby, G. et al. Candidate-gene screening and association analysis at the autism-susceptibility locus on chromosome 16p: evidence of association at GRIN2A and ABAT. Am. J. Hum. Genet. 76, 950–966 (2005).

  16. 16

    Martin, C.L. et al. Cytogenetic and molecular characterization of A2BP1/FOX1 as a candidate gene for autism. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 144B, 869–876 (2007).

  17. 17

    Sebat, J. et al. Strong association of de novo copy number mutations with autism. Science 316, 445–449 (2007).

  18. 18

    Kiehl, T.R., Shibata, H., Vo, T., Huynh, D.P. & Pulst, S.M. Identification and expression of a mouse ortholog of A2BP1. Mamm. Genome 12, 595–601 (2001).

  19. 19

    Ponthier, J.L. et al. Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16. J. Biol. Chem. 281, 12468–12474 (2006).

  20. 20

    Yeo, G.W. et al. Alternative splicing events identified in human embryonic stem cells and neural progenitors. PLOS Comput. Biol. 3, 1951–1967 (2007).

  21. 21

    McKee, A.E. et al. A genome-wide in situ hybridization map of RNA-binding proteins reveals anatomically restricted expression in the developing mouse brain. BMC Dev. Biol. 5, 14 (2005).

  22. 22

    Kim, K.K., Adelstein, R.S. & Kawamoto, S. Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J. Biol. Chem. 284, 31052–31061 (2009).

  23. 23

    Kim, K.K., Kim, Y.C., Adelstein, R.S. & Kawamoto, S. Fox-3 and PSF interact to activate neural cell-specific alternative splicing. Nucleic Acids Res. (2011).

  24. 24

    Lee, J.A., Tang, Z.Z. & Black, D.L. An inducible change in Fox-1/A2BP1 splicing modulates the alternative splicing of downstream neuronal target exons. Genes Dev. 23, 2284–2293 (2009).

  25. 25

    Damianov, A. & Black, D.L. Autoregulation of Fox protein expression to produce dominant negative splicing factors. RNA 16, 405–416 (2010).

  26. 26

    Tang, Z.Z., Zheng, S., Nikolic, J. & Black, D.L. Developmental control of CaV1.2 L-type calcium channel splicing by Fox proteins. Mol. Cell. Biol. 29, 4757–4765 (2009).

  27. 27

    Tronche, F. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999).

  28. 28

    Graus-Porta, D. et al. Beta1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex. Neuron 31, 367–379 (2001).

  29. 29

    Herrera, D.G. & Robertson, H.A. Activation of c-fos in the brain. Prog. Neurobiol. 50, 83–107 (1996).

  30. 30

    Bertram, E.H. Temporal lobe epilepsy: where do the seizures really begin? Epilepsy Behav. 14 (Suppl 1), 32–37 (2009).

  31. 31

    Ben-Ari, Y. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, 375–403 (1985).

  32. 32

    Racine, R.J. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281–294 (1972).

  33. 33

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

  34. 34

    König, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

  35. 35

    Zhang, C. et al. Integrative modeling defines the Nova splicing-regulatory network and its combinatorial controls. Science 329, 439–443 (2010).

  36. 36

    Delorenzo, R.J., Sun, D.A. & Deshpande, L.S. Cellular mechanisms underlying acquired epilepsy: the calcium hypothesis of the induction and maintainance of epilepsy. Pharmacol. Ther. 105, 229–266 (2005).

  37. 37

    Mulley, J.C., Scheffer, I.E., Petrou, S. & Berkovic, S.F. Channelopathies as a genetic cause of epilepsy. Curr. Opin. Neurol. 16, 171–176 (2003).

  38. 38

    Chapman, A.G., Woodburn, V.L., Woodruff, G.N. & Meldrum, B.S. Anticonvulsant effect of reduced NMDA receptor expression in audiogenic DBA/2 mice. Epilepsy Res. 26, 25–35 (1996).

  39. 39

    Zapata, A. et al. Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies. Brain Res. 745, 114–120 (1997).

  40. 40

    Papale, L.A. et al. Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice. Hum. Mol. Genet. 18, 1633–1641 (2009).

  41. 41

    Corradini, I., Verderio, C., Sala, M., Wilson, M.C. & Matteoli, M. SNAP-25 in neuropsychiatric disorders. Ann. NY Acad. Sci. 1152, 93–99 (2009).

  42. 42

    Johansson, J.U. et al. An ancient duplication of exon 5 in the Snap25 gene is required for complex neuronal development/function. PLoS Genet. 4, e1000278 (2008).

  43. 43

    Liu, P., Jenkins, N.A. & Copeland, N.G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003).

  44. 44

    Farley, F.W., Soriano, P., Steffen, L.S. & Dymecki, S.M. Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28, 106–110 (2000).

  45. 45

    Grabowski, P.J. Splicing-active nuclear extracts from rat brain. Methods 37, 323–330 (2005).

  46. 46

    Maguire, J., Ferando, I., Simonsen, C. & Mody, I. Excitability changes related to GABAA receptor plasticity during pregnancy. J. Neurosci. 29, 9592–9601 (2009).

  47. 47

    Sugnet, C.W. et al. Unusual intron conservation near tissue-regulated exons found by splicing microarrays. PLOS Comput. Biol. 2, e4 (2006).

  48. 48

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

Download references


This work was done in collaboration with X.-D. Fu (University of California, San Diego). We thank N. Copeland (Institute of Molecular and Cell Biology, Singapore) for the recombineering vectors and bacterial strains used for generating the transgenic Rbfox1 mice and J.P. Donahue for his help with the microarray analyses. D. Geschwind, K. Martin and T. Nilsen gave us helpful comments on the manuscript. This work was supported in part by US National Institutes of Health Grants R01 GM049369 to X.D.F., R37 NS30549 and R01 MH076994 to I.M., R01 GM084317 to M.A. and D.L.B., and R01 GM49662 to D.L.B. D.L.B. is an Investigator of the Howard Hughes Medical Institute.

Author information

Project conception: D.L.B., P.S. and L.T.G. Creation of transgenic mice: P.S. Phenotypic analysis, histology, immunofluorescence and RT-PCR studies: L.T.G. Behavioral seizure analyses: J.M., L.T.G. and I.M. Electrophysiology: J.M. and I.M. iCLIP study: A.D. and C.-H.L. Microarray studies: L.S., L.T.G. and M.A. Manuscript preparation: L.T.G. and D.L.B.

Correspondence to Douglas L Black.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–3. (PDF 2050 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading