Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.     

Regulation of coiled-coil assembly in tropomyosins

Tropomyosins (TMs) are a family of actin filament-binding proteins. They consist of nearly 100% alpha-helix and assemble into parallel coiled-coil dimers. In vertebrates, TMs are encoded by four genes that give rise to at least 17 distinct isoforms through the use of alternative RNA splicing and alternative promoters. We have studied various aspects of the coiled-coil interactions among muscle and nonmuscle isoforms by the use of transfection of epitope-tagged constructs, followed by immunoprecipitation, SDS-PAGE, and Western blot analyses. For coiled-coil interactions between high-molecular-weight isoforms (284 amino acids), the information for homo- versus heterodimerization is contained in large part within the alternatively spliced exons of nonmuscle and muscle (skeletal and smooth) isoforms. Furthermore, sequences located in alternatively spliced exons encoding amino acids 39-80 (exons 2a/2b), amino acids 189-213 (exons 6a/6b), and amino acids 258-284 (exons 9a/9d) are critical for the selective formation of homo- versus heterodimers. Among low-molecular-weight isoforms (248 amino acids), TM-4 and TM-5 can form either homodimers or heterodimers. The trigger sequence (amino acids 190-202) is required for homodimerization of TM-4, but not heterodimerization of TM-4 with TM-5. How the dimeric state of TMs might play a role in their cellular localization and function is discussed.     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.     

Four fibroblast tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene via alternative RNA splicing and the use of two promoters

cDNA clones encoding four rat tropomyosin isoforms, termed TM-2, TM-3, TM-5a, and TM-5b, were isolated and characterized. All are derived from the alpha-tropomyosin gene via alternative RNA processing and the use of two alternate promoters. The cDNA sequences predict that TM-2 and TM-3 both contain 284 amino acids and differ from each other only at an internal region of the protein from amino acids 189 through 213, due to alternative splicing of exons 6a and 6b. TM-5a and TM-5b both contain 248 amino acids and differ from each other only at an internal exon encoding amino acids 153 through 177, also due to alternative splicing of exons 6a and 6b. The differences in the amino acid sequence encoded by these alternate exons affects the theoretical actin-binding pattern of the tropomyosins, such that TM-5b is expected to bind actin with greater affinity than TM-5a. TM-2 and TM-3 are transcribed from the upstream promoter, and TM-5a and TM-5b are transcribed from an internal promoter. In addition, all four isoforms contain the identical COOH-terminal coding region. RNA protection analyses revealed that the mRNA for each isoform is expressed in a number of different tissues and cell types, although the expression of some isoforms is restricted to particular cell types. Furthermore, the expression of mRNA encoding these isoforms was found to be altered in a number of different virally transformed cell lines. The changes in the expression of tropomyosin mRNAs in transformed cells reflect changes in the relative use of the two promoters, as well as the relative use of alternatively spliced exons 6a and 6b.     

Differential localization of tropomyosin isoforms in cultured nonmuscle cells

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Nucleotide sequence of cDNA for nonmuscle tropomyosin 5 of mouse fibroblast

A cDNA clone for mouse fibroblast tropomyosin (TM) 5 was obtained from a cDNA library using human TM pseudogene as a probe. Sequence analysis of the clone revealed that the deduced amino acid sequence is different only at the 4th position from the human counterpart and that both the 5' and 3' untranslated regions are highly conserved.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

Structural and functional properties of the non-muscle tropomyosins

Summary The non-muscle tropomyosins (TMs), isolated from such tissues as platelets, brain and thyroid, are structurally very similar to the muscle TMs, being composed of two highly -helical subunits wound around each other to form a rod-like molecule. The non-muscle TMs are shorter than the muscle TMs; sequence analysis demonstrates that each subunit of equine platelet TM consists of 247 amino acids, 37 fewer than for skeletal muscle TM. The major differences in sequence between platelet and skeletal muscle TM are found near the amino and carboxyl terminal ends of the proteins. Probably as the result of such alterations, the non-muscle TMs aggregate in a linear end-to-end manner much more weakly than do the muscle TMs. Since end-to-end interactions are responsible for the highly cooperative manner in which TM binds to actin, the non-muscle TMs have a lower affinity for actin filaments than do the muscle TMs. However, the attachment of other proteins to actin (e.g. the Tn-I subunit of skeletal muscle troponin or the S-1 subfragment of skeletal muscle myosin) can increase the affinity of actin filaments for non-muscle TM. The non-muscle TMs interact functionally with the Tn-I component of skeletal muscle troponin to inhibit the ATPase activity of muscle actomyosin and with whole troponin to regulate the muscle actomyosin ATPase in a Ca⁺⁺-dependent manner, even though one of the binding sites for troponin on skeletal TM is missing in non-muscle TM. A novel actomyosin regulatory system can be produced using Tn-I, calmodulin and non-muscle TM; in this case inhibition is released when the non-muscle TM detaches from the actin filament in the presence of Ca⁺⁺. Although it has not yet been demonstrated that the non-muscle TMs participate in a Ca⁺⁺-dependent contractile regulatory system in vivo it does appear that they are associated with actin filaments in vivo.     

Characterization of a Drosophila cDNA clone that encodes a 252-amino acid non-muscle tropomyosin isoform

We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.     

Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855 bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former.     

Isolation and sequence of a cDNA clone that contains the entire coding region for chicken smooth-muscle alpha-tropomyosin

We have constructed a cDNA-expression library of approximately 100,000 members from embryonic chicken smooth-muscle mRNA using the plasmid-expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, we have identified and isolated clones encoding smooth-muscle tropomyosin. Plasmid pSMT-10 (approximately 1100 base pairs) was found to hybrid-select mRNA for smooth-muscle alpha-tropomyosin. DNA-sequence analysis revealed that pSMT-10 contained the entire coding region for alpha-tropomyosin and portions of the 5'- and 3'-untranslated regions. Comparison of the derived amino acid sequence of smooth-muscle alpha-tropomyosin with known skeletal-muscle (rabbit and chicken) and platelet (equine) sequences revealed extensive homology between the various proteins. The smooth-muscle tropomyosin shows the greatest sequence divergence from the skeletal-muscle tropomyosins at the COOH-terminal region. In contrast, the smooth-muscle tropomyosin is most homologous to the platelet tropomyosin at the COOH-terminal end. The relationship of the various tropomyosin sequences to function (e.g. interactions with troponin) are considered.     

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.     

Fish fast skeletal muscle tropomyosins show species-specific thermal stability

Tropomyosin (TM) was isolated from the fast skeletal muscle of six fish species, whose amino acid sequences of this protein have already been revealed. The thermal stability of these TMs was measured by differential scanning calorimetry (DSC) and circular dichroism (CD), while the molecular weights were measured by mass spectrometry. The results showed clear differences in thermostability among these fish TMs, though the identity of amino acid sequences was more than 93.3%. Therefore, only a few amino acid substitutions could affect the overall stability of the TM molecule. Especially, several residues located on the molecular surface were considered to be responsible for such stability difference. In contrast, the molecular weights of these TMs as measured by mass spectrometry were higher than those calculated from amino acid composition, suggesting the presence of post-translational modification(s) which could also affect their thermal stability.