The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain - PubMed (original) (raw)

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

Lauren T Gehman et al. Nat Genet. 2011.

Abstract

The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts, but its role in neuronal physiology is not clear. 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.

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Conflict of interest statement

Competing interests statement: The authors declare that they have no competing financial interests.

Figures

Figure 1

Figure 1. Rbfox1−/− brains lack Rbfox1 protein expression but possess normal morphology

(a) Confocal immunofluorescence microscopy on coronal sections of wild-type (WT) dentate gryus probed for Rbfox1 (green) and Rbfox2 (red); overlayed images are shown in the far right panels. Arrows point to cells expressing only Rbfox1; arrowheads point to cells expressing only Rbfox2. Scale bar, 100 μm. (b) Immunoblot analysis of Rbfox1 and Rbfox2 in nuclear lysates isolated from WT, Rbfox1+/− and Rbfox1−/− brains. U1-70K was used as a loading control for total nuclear protein. Below each gel is the amount of Rbfox1 or Rbfox2 protein in each sample as a percentage of WT, normalized by U1-70K expression. (c) Confocal immunofluorescence microscopy on coronal sections of WT and Rbfox1−/− dentate gyrus probed for Rbfox1 (green) and Rbfox2 (red) expression. Overlayed images are shown in the far right panels. Scale bar, 100 μm. (d) Representative Nissl stain of WT and _Rbfox1−/−_hippocampus at 1 mo of age showing normal gross morphology. Scale bar, 0.5 mm. Abbreviations: CA1/CA3, pyramidal layers of the hippocampus; DG, dentate gyrus.

Figure 2

Figure 2. Rbfox1−/− brains are epileptic and hyperexcitable

(a) c-Fos immunostaining on WT and Rbfox1−/− coronal brain sections 1 h after a spontaneous seizure in the _Rbfox1loxP/loxP/Nestin-Cre+/−_mouse. Relevant brain areas are indicated. Abbreviations: amyg, amygdala; CA1/CA3, pyramidal cell layers of the hippocampus; DG, dentate gyrus; ent, entorhinal cortex; II-III indicate layers of the cerebral cortex. Scale bar, 2 mm. (b) Progression of behavioral changes after systemic KA administration (20mg/kg, i.p.) in WT, Rbfox1loxP/loxP/Nestin-Cre+/−, and _Rbfox2loxP/loxP/Nestin-Cre+/−_mice over a 2 h observation period. Seizures were scored on the Racine Scale as described and data shown are mean scores. Error bars, s.e.m. (c) Representative fEPSP traces from individual electrophysiological recordings in WT and Rbfox1−/− dentate gyrus. (d) Average synaptic I/O curves in WT and Rbfox1−/− dentate fit with a Boltzmann function (solid lines). Circles are grand-averaged scores; error bars, s.e.m. _W_50, stimulus width that elicits 50% of the maximum response; k, slope factor. n = 3 mice, 16–20 slices per experimental group.

Figure 3

Figure 3. Rbfox1−/− brain exhibits splicing changes in transcripts affecting synaptic function and neuronal excitation

(a) Denaturing gel electrophoresis of RT-PCR products for three Rbfox1-dependent exons. Above each gel is a schematic depicting the alternative exon (horizontal black box) and the relative location of (U)GCAUG binding sites (yellow boxes) in the flanking introns (thin horizontal lines). Shown below each gel is a graph quantifying the mean percentage of alternative exon inclusion (% Ex In, PSI) in WT (black bars), Rbfox1 knockout (KO; red bars), and Rbfox2 KO (blue bars) brain. (b) A schematic showing the Snap25 mutually exclusive exon pair, 5a (horizontal black box) and 5b (horizontal gray box), plus the intervening intron and the promixal 500 nt of the adjacent introns. Yellow boxes represent (U)GCAUG motifs. The distribution of Rbfox1 iCLIP reads (horizontal green bars) is shown above. A histogram displaying the conservation of this region among 30 vertebrate species, as determined by phastCons (

http://genome.ucsc.edu

), is shown below. A score of 1 indicates 100% identity among all species at that nt position. Chromosomal location in nt is shown above the iCLIP data. At the bottom, the RT-PCR assay and quantification of exons 5a and 5b splicing is shown, with the inclusion of the downstream exon plotted. For (a) and (c), error bars represent s.e.m.; n = 3. *P<0.05 and n.s. = not significant by paired, one-tailed Student’s t test. Exact P values are shown in Table 1.

Comment in

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