In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for tissue-dependent alternative splicing of two nonmuscle MHC II (NMHC II) genes, NMHC II-B and NMHC II-C. Our past study using cultured cells revealed that an RNA binding protein family, the Rbfox family, plays a role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. Rbfox proteins contain a single conserved RNA recognition motif in the central region of the molecule and bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Biochemical analyses of mouse brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox3 expression rather than with that of Rbfox-1 or Rbfox2 expression, although Rbfox3 and some of the isoforms of Rbfox1 and 2 are similarly capable of enhancing the neuron-specific exon B1 splicing when they are over-expressed in cultured cells. These observations suggest that Rbfox3 plays a more physiologically relevant role in neuron-specific splicing of NMHC II-B mRNA in vivo. To understand a mechanism for Rbfox3-mediated regulation of alternative splicing, we searched for nuclear factor(s) which interact with Rbfox3. We identified PTB- associated splicing factor (PSF, also called SFPQ) as an interacting protein with Rbfox3 by antibody affinity-isolation of Rbfox3-containing complexes from mouse brain extracts followed by mass spectrometry. The C-terminal region of Rbfox3 directly binds to the N-terminal region of PSF. In cultured cells, enhancement of B1 inclusion by Rbfox3 depends on the presence of PSF. Rbfox3 is recruited to the UGCAUG element downstream of B1 in the endogenous NMHC II-B transcript in a PSF-dependent manner. PSF enhances B1 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Therefore, PSF functions as an essential co-activator of Rbfox3. Rbfox3 and PSF interaction is an integral part of the mechanism by which Rbfox proteins regulate activation of alternative exons via a downstream intronic enhancer. We are now extending our research to study muscle-specific skipping of the alternative exon C1 in NMHC II-C mRNAs. Mouse myogenic cell lines C2C12 and G8 provide useful models in which the expression level of the C1-excluded NMHC II-C mRNA splice variant over the C1-included variant increases during differentiation from myoblasts to multinucleated myotubes. Using minigene constructs containing the C1 exon and flanking introns and exons, a proximal UGCAUG element upstream of C1 was found to be important for C1 exclusion in mRNAs, although there are multiple UGCAUG elements in both upstream and downstream introns of C1. Overexpression of either Rbfox1 and 2 in myoblasts enhances C1 exclusion via the upstream UGCAUG. This is in contrast with Rbfox-activated inclusion of B1 which requires the downstream UGCAUG element. These results suggest that Rbfox proteins have dual functions for alternative splicing: to either activate or to repress an alternative exon, presumably by different mechanisms.
