RBFOX1

Abstract: Cytogenetic imbalances are increasingly being realized as causes of autism Here, we report a de novo translocation between the short arms of chromosomes 15 and 16 in a female with autism, epilepsy, and global developmental delay FISH analysis identified a cryptic deletion of approximately 160 kb at the boundary of the first exon and first intron of the 17 Mb ataxin-2 binding protein-1 (A2BP1) gene, also called FOX1 Quantitative real time PCR (Q-PCR) analysis verified a deletion of exon 1 in the 5' promoter region of the A2BP1 gene Reverse transcription PCR (qRT-PCR) showed reduced mRNA expression in the individual's lymphocytes, demonstrating the functional consequence of the deletion A2BP1 codes for a brain-expressed RNA binding or splicing factor Because of emerging evidence in the role of RNA processing and gene regulation in pervasive developmental disorders, we performed further screening of A2BP1 in additional individuals with autism from the Autism Genetics Resource Exchange (AGRE) collection Twenty-seven SNPs were genotyped across A2BP1 in 206 parent-child trios and two regions showed association at P < or = 0008 level No additional deletions or clear mutations were identified in 88 probands by re-sequencing of all exons and surrounding intronic regions or quantitative PCR (Q-PCR) of exon 1 Although only nominal association was observed, and no obvious causal mutations were identified, these results suggest that A2BP1 may affect susceptibility or cause autism in a subset of patients Further investigations in a larger sample may provide additional information regarding the involvement of this gene in the autistic phenotype

Abstract: Alternative pre-mRNA splicing is an important mechanism for regulating gene expression that contributes greatly to proteomic diversity in eukaryotes (Black 2003; Blencowe 2006; Nilsen and Graveley 2010). Changes in exon inclusion or splice site usage can substantially alter the expression or function of the encoded protein. Alternative splicing is especially prevalent in the mammalian nervous system, where it controls aspects of neural tube patterning, synaptogenesis, and the regulation of membrane physiology, among other important processes (Lipscombe 2005; Licatalosi and Darnell 2006; Li et al. 2007). The choice of splicing pattern is generally controlled by trans-acting RNA-binding proteins that bind to cis-acting elements in the pre-mRNA to enhance or silence particular splicing events (Black 2003; Matlin et al. 2005; Chen and Manley 2009; Nilsen and Graveley 2010). These RNA-binding proteins can be expressed in a temporal- or tissue-specific manner to alter the splicing of a defined set of transcripts. Some of these splicing regulators have been shown to play important roles in the developing and adult mammalian brain (Jensen et al. 2000; Lukong and Richard 2008; Calarco et al. 2009; Yano et al. 2010; Gehman et al. 2011; Raj et al. 2011; Zheng et al. 2012). In mammals, the RNA-binding Fox (Rbfox) family of splicing regulators is comprised of three members: Rbfox1 (Fox-1 or A2BP1), Rbfox2 (Fox-2 or RBM9), and Rbfox3 (Fox-3, HRNBP3, or NeuN) (Kuroyanagi 2009). Each Fox protein has a single central RNA recognition motif (RRM) RNA-binding domain that recognizes the sequence (U)GCAUG found within introns flanking alternative exons (Jin et al. 2003; Auweter et al. 2006; Ponthier et al. 2006). The position of the (U)GCAUG motif with respect to the alternative exon dictates the effect of the Rbfox proteins on splicing. A motif located downstream from the alternative exon generally promotes Rbfox-dependent exon inclusion, whereas an upstream motif will usually repress inclusion (Huh and Hynes 1994; Modafferi and Black 1997; Jin et al. 2003; Nakahata and Kawamoto 2005; Underwood et al. 2005; Zhang et al. 2008; Kuroyanagi 2009; Yeo et al. 2009). The three mouse Rbfox paralogs show a high degree of sequence conservation, especially within the RNA-binding domain, which is identical between Rbfox1 and Rbfox2 and only slightly altered in Rbfox3 (94% amino acid identity). The N-terminal and C-terminal domains are less similar between the proteins, presumably allowing for different protein–protein interactions. All three Rbfox family members are highly expressed in most neurons of the mature brain, where they regulate the splicing of neuronal transcripts (McKee et al. 2005; Nakahata and Kawamoto 2005; Underwood et al. 2005; Kim et al. 2009; Tang et al. 2009; Hammock and Levitt 2011). Rbfox1 and Rbfox2 have been shown to control a shared set of neuronal-specific target exons, including exon N30 of nonmuscle myosin heavy chain II-B (NMHC-B), exon N1 of c-src, and exons 9* and 33 of the L-type calcium channel Cav1.2 (Nakahata and Kawamoto 2005; Underwood et al. 2005; Tang et al. 2009). The individual Rbfox family members show differing patterns of expression. Rbfox1 is expressed in neurons, heart, and muscle, while Rbfox3 is limited to neurons (Wolf et al. 1996; Jin et al. 2003; McKee et al. 2005; Underwood et al. 2005; Kim et al. 2009; Damianov and Black 2010). Rbfox2 is expressed in these tissues as well as other cell types, including the embryo, hematopoietic cells, and embryonic stem cells (ESCs) (Underwood et al. 2005; Ponthier et al. 2006; Yeo et al. 2007). Thus, although the Rbfox proteins can regulate many of the same target exons when ectopically expressed, their in vivo targets may differ due to the variable expression of each protein. For example, Rbfox2 controls the developmental-specific splicing of exons in fibroblast growth factor receptor 2 (FGFR2), erythrocyte protein 4.1R, and STE20-like kinase in cells where the other proteins are absent (Baraniak et al. 2006; Ponthier et al. 2006; Yang et al. 2008; Yeo et al. 2009). Rbfox2 is clearly important for splicing regulation during embryonic growth and development, but its role in the brain is less clear. Defects in alternative splicing can lead to neurological and neuromuscular disease, such as frontotemporal dementia and myotonic dystrophy (Faustino and Cooper 2003; Licatalosi and Darnell 2006; Cooper et al. 2009). The Rbfox proteins have also been linked to neurological conditions. Human mutations in the RBFOX1 (A2BP1) gene can lead to severe disorders, including mental retardation, epilepsy, and autism spectrum disorder (Bhalla et al. 2004; Barnby et al. 2005; Martin et al. 2007; Sebat et al. 2007; Voineagu et al. 2011). Moreover, human RBFOX1 was first identified through an interaction with Ataxin-2, the protein mutated in spinocerebellar ataxia type II (SCAII), and RBFOX2 was later shown to interact with Ataxin-1, which is mutated in SCAI patients (Shibata et al. 2000; Lim et al. 2006). These results imply a role for Rbfox proteins in cerebellar function. We recently showed that deletion of Rbfox1 results in increased neuronal excitation in the hippocampus and seizures in the mouse, in keeping with its regulation of many gene products important for synaptic transmission (Gehman et al. 2011). Rbfox1 mutation did not lead to obvious cerebellar defects. Interestingly, deletion of Rbfox2 did not produce the same seizure phenotype as Rbfox1 deletion. Thus, while the Rbfox proteins share some target exons in the brain, they are not fully redundant in their functions. To better understand the roles of Rbfox-mediated splicing regulation in the brain, we created mice with tissue- and cell type-specific deletions of one or more Rbfox proteins. We found that CNS-specific deletion of Rbfox2 results in impaired cerebellar development and additional neurological phenotypes, whereas postnatal deletion from cerebellar Purkinje neurons leads to marked deficits in neuronal excitability and, specifically, pacemaking. Thus, like Rbfox1, Rbfox2 is essential for the proper function of mature neural circuits, but also plays a role in brain development.

Abstract: RNA splicing plays a critical role in the programming of neuronal differentiation and, consequently, normal human neurodevelopment, and its disruption may underlie neurodevelopmental and neuropsychiatric disorders. The RNA-binding protein, fox-1 homolog (RBFOX1; also termed A2BP1 or FOX1), is a neuron-specific splicing factor predicted to regulate neuronal splicing networks clinically implicated in neurodevelopmental disease, including autism spectrum disorder (ASD), but only a few targets have been experimentally identified. We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins. Downstream alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. Several of these differentially expressed genes are further implicated in ASD and related neurodevelopmental diseases. Weighted gene co-expression network analysis demonstrates a high degree of connectivity among these disease-related genes, highlighting RBFOX1 as a key factor coordinating the regulation of both neurodevelopmentally important alternative splicing events and clinically relevant neuronal transcriptional programs in the development of human neurons.

Abstract: RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.