Alternative Splicing of Nonmuscle Myosin Heavy Chains

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Myosin II protein molecules, which consist of a pair of heavy chains (approximately 200 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 (MHCs). MHC 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 isoforms as well as changes in MHC 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 genes, NMHC-A, NMHC-B, and NMHC-C. The purpose of this research program is to understand the mechanisms underlying transcriptional regulation of NMHC-A and C genes as well as tissue-dependent regulation of alternative splicing of NMHC-B and C genes. In this report, we focus on regulation of alternative splicing of NMHC-B and C and regulation of NMHC-C transcription. The genes encoding NMHC-B and 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-B mRNAs is restricted to some neural cells. On the other hand, alternative exon C1 encoding 8 aa is excluded entirely from NMHC-C mRNAs in striated muscle, but is dominantly included in nonmuscle cell mRNAs. We have found that the intronic RNA element UGCAUG and sequence-specific RNA-binding proteins, Fox-1 family 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 elements, but not the downstream ones, are found to be important for C1 exclusion in muscle cells. We further examined the effects of exogenous expression of Fox-1 family proteins, which can bind to the UGCAUG element, during B1 and C1 splicing. 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. 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-2. We have made expression constructs encoding 24 Fox-2 isoforms, which differ in N-terminal, internal and C-terminal sequences, but share an identical RRM. Inclusion of B1 is activated by the isoforms containing a particular internal and C-terminal sequence. In contrast, exclusion of C1 can be enhanced by all isoforms. These results suggest that the domains of Fox-2 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-2 confer tissue-specificity on alternative splicing of B1 and C1. With respect to NMHC-C transcription, we have identified two alternative promoters for the mouse gene, which are utilized in a tissue-dependent manner. The promoter located more distally from the common exon 2 is utilized in a wide variety of tissues including brain and heart in adult mice, whereas the more proximal promoter is predominantly utilized in lung. Both promoters lack a TATA box and show a high GC content, similar to the single promoter found for NMHC-A and NMHC-B. Unlike NMHC-A and B, however, NMHC-C mRNAs are expressed at the very low level at early embryonic stages as well as embryonic stem cells. Consistent with this fact, many cell lines derived from tissues where the NMHC-C mRNAs are expressed in adulthood, express only very low levels of NMHC-C. Using a number of cell lines, we have found that CpG methylation and histone deacetylation play critical roles in repression of the distal promoter of the NMHCII-C gene and that Sp3 plays a role in activation of this promoter.