An inducible change in Fox-1/A2BP1 splicing modulates the alternative splicing of downstream neuronal target exons

Abstract

Neuronal depolarization and CaM kinase IV signaling alter the splicing of multiple exons in transcripts for ion channels, neurotransmitter receptors, and other synaptic proteins. These splicing changes are mediated in part by special CaM kinase-responsive RNA elements, within or adjacent to exons that are repressed in the initial phase of chronic depolarization. The splicing of many neuronal transcripts is also regulated by members of the Fox (Feminizing gene on X) protein family, and these Fox targets are also often proteins affecting synaptic activity. We show that Fox-1/Ataxin 2-Binding Protein 1 (A2BP1), a protein implicated in a variety of neurological diseases, can counteract the effects of chronic depolarization on splicing. We find that exon 19 of Fox-1 is itself repressed by depolarization. Fox-1 transcripts missing exon 19 encode a nuclear isoform of Fox-1 that progressively replaces the cytoplasmic Fox-1 isoform as cells are maintained depolarizing media. The resulting increase in nuclear Fox-1 leads to the reactivation of many Fox-1 target exons, including exon 5 of the NMDA receptor 1, that were initially repressed by the high-KCl medium. These results reveal a novel mechanism for the slow modulation of splicing as cells adapt to chronic stimuli: The subcellular localization of a splicing regulator is controlled through its own alternative splicing. Alternative pre-mRNA splicing is an important mechanism for controlling gene expression in metazoan organisms. Changes in splice site or exon usage frequently determine the function of a gene product or eliminate its expression (Black 2003;Matlin et al. 2005;Blencowe 2006). The best understood systems of splicing regulation are those modulated by cell type, where stable differences in pre-mRNA-binding protein expression determine a splicing choice. However, splicing is also dynamically regulated by external stimuli and growth conditions. Most cellular signaling pathways that alter gene transcription also alter groups of alternative exons (Shin and Manley 2004;Blaustein et al. 2007;Stamm 2008). In some cases, these responses have been traced to particular regulatory elements in the pre-mRNA, to post-translational modifications of splicing regulators, or to changes in transcriptional control. However, the mechanisms that allow the splicing machinery to respond to dynamic stimuli are not well understood.A particularly interesting regulatory mechanism in excitable cells is their response to membrane depolarization. Depolarization-induced changes in neuronal transcription are well studied and involve the activation of CaM kinase and other signaling pathways to induce transcription of both rapid primary response genes and more slowly developing secondary responses (Flavell and Greenberg 2008;Greer and Greenberg 2008). These transcriptional programs play important roles in neuronal plasticity and modulating synaptic activation. Ion channel mRNAs and other transcripts affecting membrane activity also often contain exons whose splicing responds to chronic depolar...

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

“…2A). This result is consistent with previous reports that the C-terminal region is critical for nuclear localization (Nakahata and Kawamoto 2005;Lee et al 2009). …”

“…This is consistent with our localization results. It has also been noted that some RBFOX1 isoforms lack the second half of the C-terminal domain because of frame-shifting, and therefore, they are not functional in splicing regulation (Nakahata and Kawamoto 2005;Lee et al 2009). …”

“…Therefore, some of these isoforms are expected to have different activities or to lack alternative splicing functions (Nakahata and Kawamoto 2005). Alternative splicing of RBFOX1 is regulated during neuronal depolarization, allowing it to modulate the activity of its target genes (Lee et al 2009). …”

Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

“…Several previous studies have demonstrated that depolarization of neuronal cells induced changes of splicing regulators, which in turn led to alternative splicing changes. [33][34][35][36][37][38] It is possible that the calcium-induced export of class II HDACs changes of post-translational modification of some splicing regulators, e.g., making some proteins more acetylated, which alters their function and then leads to downstream alternative splicing changes. It is very likely that multiple mechanisms exist that control calcium-mediated alternative splicing changes.…”

Neutralization of terminal differentiation in gliomagenesis

“…P19 cells were differentiated into postmitotic neuronal cells as described previously (50). Briefly, P19 cells were grown in αMEM with 10% FBS.…”