Myosin II protein molecules, which consist of a pair of heavy chains (approximately 230 kDa) and two pairs of light chains (15-28 kDa), exist in all eukaryotic cells. Together with actin filaments, they produce contractile force mediated by ATP hydrolysis. While this contractile activity is prominent in differentiated muscle tissues, it is also involved in diverse cellular motile processes such as cytokinesis, cell migration and cell adhesion in nonmuscle cells, as well as in undifferentiated muscle cells. 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 the course of muscle and nervous tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. In this report, we focus on regulation of alternative splicing of NMHC II-B and II-C. The genes encoding NMHC II-B and II-C generate alternatively spliced isoforms, which include or exclude a cassette of amino acids (aa) near the ATP-binding domain. Inclusion of alternative exon B1 (also called exon N30) encoding 10 aa in NMHC II-B mRNAs is restricted to some neural cells. On the other hand, alternative exon C1 encoding 8 aa is excluded entirely from NMHC II-C mRNAs in striated muscles, but is predominantly included in nonmuscle cell mRNAs. We have found that the intronic RNA element UGCAUG and a family of sequence-specific RNA-binding proteins, Fox-1 proteins, play a critical role in regulation of both B1 and C1 alternative splicing, but in a different manner. Minigene transfection analysis using a number of cell lines has revealed that the UGCAUG elements located in the downstream intron of B1 is essential for B1 inclusion in neural cells. Interestingly, this RNA element is also present in both the upstream and downstream introns of C1. However, only the upstream UGCAUG element, but not the downstream one, is found to be important for C1 exclusion in muscle cells. We also characterized the Fox-1 family of proteins, which can bind to the UGCAUG element. Three genes, Fox-1, Fox-2 and Fox-3, which contain highly homologous RNA recognition motifs (RRMs), belong to the Fox-1 family in mammals. Fox-1 is expressed in brain and striated muscles whereas Fox-2 is expressed in various tissues including brain and muscles. Fox-3 is expressed exclusively in brain. RT-PCR, 5 RACE and analysis of sequence databases have revealed that multiple alternative promoters and alternative splicing of pre-mRNA give rise to a number of tissue-dependent isoforms of Fox-1 and Fox-2. We have made expression constructs encoding various Fox-1 and Fox-2 isoforms as well as Fox-3, which differ in N-terminal, internal and C-terminal sequences, but share a highly homologous RRM. We have examined the effects of exogenous expression of these Fox isoforms during B1 and C1 splicing. Inclusion of B1 can be activated by Fox-3 and the particular isoforms of Fox-1 and Fox-2, which share the homologous internal and C-terminal sequences with Fox3. These isoforms are preferentially expressed in brain but not in striated muscles. In contrast, exclusion of C1 can be enhanced by all Fox isoforms examined, regardless of poor homology in N-terminal and C-terminal sequences. These results suggest that the domains of Fox isoforms responsible for B1 inclusion and C1 exclusion are different. Further, this study suggests that a different distribution of the same splicing regulatory element in pre-mRNAs as well as isoform diversity of Fox-1 family proteins confer tissue-specificity on alternative splicing of B1 and C1. In addition to the above findings, during the course of characterization of Fox-3, we found that NeuN, which is a widely used marker of post-mitotic neural cell nuclei as revealed by a specific monoclonal antibody for nearly two decades, is the Fox-3 gene product.
