NeuN (neuronal nuclei) is a neuron-specific nuclear protein which is identified by immunoreactivity with a monoclonal antibody, anti-NeuN. Anti-NeuN has been used widely as a reliable tool to detect most postmitotic neuronal cell types in neuroscience, developmental biology, and stem cell research fields as well as diagnostic histopathology. To date, however, the identity of its antigen, NeuN itself, has been unknown. Here, we identify NeuN as the Fox-3 gene product by providing the following evidence: 1) Mass spectrometry analysis of anti-NeuN immunoreactive protein yields the Fox-3 amino acid sequence. 2) Recombinant Fox-3 is recognized by anti-NeuN. 3) Short hairpin RNAs targeting Fox-3 mRNA down-regulate NeuN expression. 4) Fox-3 expression is restricted to neural tissues. 5) Anti-Fox-3 immunostaining and anti-NeuN immunostaining overlap completely in neuronal nuclei. We also show that a protein crossreactive with anti-NeuN is the synaptic vesicle protein, synapsin I. Anti-NeuN recognizes synapsin I in immunoblots with one order of magnitude lower affinity than Fox-3, and does not recognize synapsin I using immunohistology. Fox-3 (also called hexaribonucleotide-binding protein 3 and D11Bwg0517e) contains an RNA recognition motif and is classified as a member of the Fox-1 gene family that binds specifically to an RNA element, UGCAUG. We demonstrate that Fox-3 functions as a splicing regulator using neural cell-specific alternative splicing of the non-muscle myosin heavy chain II-B pre-mRNA as a model. Identification of NeuN as Fox-3 clarifies an important element of neurobiology research. Mullen et al. (1) have reported a monoclonal antibody (mAb)2 , which was generated using brain cell nuclei as antigens. This mAb recognizes 2-3 protein bands with apparent molecular masses of 46 -48 kDa following SDS-PAGE that are expressed in neuronal tissues. Intensive immunohistochemical analyses using embryonic and adult murine tissues have demonstrated that this mAb stains exclusively neuronal cells in the central and peripheral nervous systems, especially postmitotic and differentiating neurons, as well as terminally differentiated neurons. The mAb staining is localized primarily to nuclei. Thus, the authors named the antigen recognized by this mAb NeuN for "Neuronal Nuclei." The original study and subsequent studies by others have shown that anti-NeuN stains most types of neurons throughout the nervous system with few exceptions (1, 2). In no case has NeuN expression been observed in glial cells. This mAb can detect the NeuN antigen in a wide range of vertebrate species, including mammals, birds, and amphibians. Because of its high specificity for postmitotic neurons, its broad specificity for most types of neuronal cells, and its cross-reactivity with multiple species, anti-NeuN has gained widespread acceptance as a reliable tool to detect neuronal cells in neuroscience and developmental biology research and in diagnostic histopathology for neural diseases and tumors (3-10). More recently, it has also been used to mo...

Rbfox3 is required to promote neuronal differentiation of postmitotic neurons through Numb alternative splicing.

Fox-1 family (Fox) proteins, which consist of Fox-1 (A2BP1), Fox-2 (Rbm9) and Fox-3 (NeuN) in mammals, bind to the RNA element UGCAUG and regulate alternative pre-mRNA splicing. However the mechanisms for Fox-regulated splicing are largely unknown. We analyzed the expression pattern of the three Fox proteins as well as neural cell-specific alternative splicing of a cassette exon N30 of nonmuscle myosin heavy chain (NMHC) II-B in the mouse central nervous system. Histological and biochemical analyses following fluorescence-activated cell sorting demonstrate a positive correlation of N30 inclusion and Fox-3 expression. Further, we identified polypyrimidine tract binding protein-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. In cultured cells, enhancement of N30 inclusion by Fox-3 depends on the presence of PSF. PSF enhances N30 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of N30 in the endogenous NMHC II-B transcript in a PSF-dependent manner. This study is the first to identify PSF as a coactivator of Fox proteins and provides evidence that the Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.