Alternative splicing regulation of APP exon 7 by RBFox proteins Shafiul Alam a , Hitoshi Suzuki a,b,⇑ , Toshifumi Tsukahara a,⇑ a School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan b Centre for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan article info Article history: Received 21 June 2014 Received in revised form 18 July 2014 Accepted 1 August 2014 Available online 11 August 2014 Keywords: Alternative splicing RBFox1 Amyloid precursor protein APP695 isoform Cis-element RNA-binding protein abstract RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer’s disease, and therefore we examined APP alter- native splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox pro- teins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 contain- ing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Alternative pre-mRNA splicing is an important genetic event involved in generating several transcripts from a single gene and, thereby, creating protein diversity. The functional consequence of this influential mechanism is that functionally distinct isoforms are produced in different cell types or during development (Blencowe et al., 2009; Calarco et al., 2011; Wang et al., 2008), regulating cellular differentiation and development of biological systems. Alternative splicing mechanisms during neuronal differ- entiation are very important to understand neuronal cell differen- tiation and development (Yeo et al., 2007). Several splicing regulators, including neural polypyrimidine tract-binding protein (nPTB or PTBP2), neuro-oncological ventral antigen 1 (Nova1), and RNA-binding protein Fox-1 (RBFox1, known as A2BP1 or Fox-1), have been implicated in neural-specific alternative splicing (Coutinho-Mansfield et al., 2007; Ule et al., 2006; Zhang et al., 2008). However, a more extensive investigation on the regulation of alternative splicing during neuronal differentiation is required to determine the underlying mechanism. RBFox1 is a neuron- and muscle-specific splicing factor that has both positive and negative regulatory effects on alternative splic- ing (Jin et al., 2003). It encompasses an RNA recognition motif (RRM)-type RNA-binding domain that exerts its activity by binding specifically to a (U)GCAUG sequence in pre-mRNA (Jin et al., 2003). (U)GCAUG sequences located in introns adjacent to neuron- and/or muscle-specific exons have been identified (Underwood et al., 2005). As shown by several groups, this sequence, when located in introns downstream of alternative exons, functions as a splicing enhancer to induce exon inclusion. It is suggested that intronic (U)GCAUG sequences upstream of regulated exons generally repress exon inclusion (Underwood et al., 2005), although this locational relationship may not be so strong (Zhang et al., 2008). Nevertheless, an upstream intronic (U)GCAUG sequence causes skipping of the alternative exon, F1c, and blocks formation of the prespliceosomal early complex (E-complex) (Fukumura et al., 2007). RBFox1 is a tissue-specific splicing factor; therefore, it is a prime candidate for understanding the underlying mechanism of alternative splicing regulation during neuronal differentiation. http://dx.doi.org/10.1016/j.neuint.2014.08.001 0197-0186/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Address: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan. Tel.: +81 761 51 1643; fax: +81 761 51 1455 (H. Suzuki). Tel.: +81 761 51 1640; fax: +81 761 51 1149 (T. Tsukahara). E-mail addresses: shafiula@jaist.ac.jp (S. Alam), suzuki-h@jaist.ac.jp (H. Suzuki), tukahara@jaist.ac.jp (T. Tsukahara). Neurochemistry International 78 (2014) 7–17 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci