The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

Abstract

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific fdr nonmuscle cells and different ypes of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of Si nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat a-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat a-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing.Tropomyosin (TM) plays an important role in the regulation of contractility of muscle and nonmuscle cells. This protein forms a coiled-coil dimer and exists in several different forms (a, 1, and nonmuscle-cytoplasmic) with apparent molecular weights ranging from 30,000 to 50,000. In striated muscles, the coiled-coil dimers form head-to-tail polymers that lie in the major groove of the thin filament, with each dimer spanning seven actin molecules (63). In striated and possibly in smooth muscle cells, TM plays a fundamental role in regulating the Ca2" activation of the contractile apparatus through its interaction with actin and the Ca2"-regulatory proteins troponin and caldesmon, respectively (see reference 39). However, the precise function of TM in nonmuscle cells remains unknown, although it might play a role in determining the architecture of the cell and modulate its contractile (16) and motile (32) activities.The different TM isoforms have a tissue-and developmental stage-specific pattern of expression. Skeletal muscle is known to contain the a and m subunits assembled into homoand heterodimers that are present in variable proportions depending on the fiber type (18-20, 59, 62). These two subunits are 284 amino acids (aa) long and highly conserved in sequence (38,64,67). In the adult myo...

Gene expression changes associated with fibronectin-induced cardiac myocyte hypertrophy

“…These include the fetal isoforms ␣SkA (Acta1) and cardiac ␤MHC (Myh7), well known to be upregulated in cardiac hypertrophy, as well as myosin light chains, cardiac myosin-binding protein C, troponin C and T, and tropomyosin genes (Supplemental Table S1). The tropomyosin genes are regulated by alternative splicing in a tissue-specific manner (57), and the RAE230A array probe sets can distinguish some of the alternatively spliced isoforms. For example, probe set 1370287_a_at recognizes the smooth muscle-specific exon 9d and showed an expression level of 1,157 and induction of 1.3-fold by FN, whereas probe set 1368724_a_at recognizes the striated muscle-specific exon 9b and showed an expression level of 16,414 and induction of 2.1-fold by FN in cardiac myocytes.…”

Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

“…The orientation of the clones was determined by restriction digestion with PstI and the sequence of clones in the correct orientation was confirmed. Northern-blot analysis was carried out using α-tropomyosin, SM22α and β-actin cDNA probes as described previously [12,34,35].…”

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

“…Whereas the smooth muscle rich tissues such as bladder and aorta showed nearly complete exon 2 inclusion and the striated muscles showed full inclusion of exon 3, a number of other tissues with little smooth muscle content, such as liver and spleen, showed significant percentages of exon 2 inclusion. Notably, these tissues had very low levels of ␣TM expression, which had not been detectable using nuclease protection or Northern blot (18). Subsequent analyses of GTM3 transgene splicing indicated that bladder and aorta provided a robust regulatory environment that was able to support significant levels of transgene exon skipping in all mouse lines, even with high levels of transgene expression.…”

“…This had previously been surveyed qualitatively by nuclease protection and was restricted primarily to those tissues with high levels of expression (18,19).…”

“…1). Exons 2 and 3 are spliced in a mutually exclusive manner, with predominant inclusion of exon 2 only in smooth muscle tissues, whereas exon 3 is used almost exclusively in striated muscles (cardiac and skeletal) and most other tissues where ␣TM expression could be observed (18,19), including proliferating VSMCs (20). Likewise, transfection of minigene constructs into almost all cell types resulted in predominant or exclusive inclusion of exon 3 (10).…”