Structure and evolution of the alternatively spliced fast troponin T isoform gene.

The vertebrate fast skeletal muscle troponin T gene, TnTf, produces a complexity of isoforms through differential mRNA splicing. The mechanisms that regulate splicing and the physiological significance of TnTf isoforms are poorly understood. To investigate these questions, we have determined the complete sequence structure of the quail TnTf gene, and we have characterized the developmental expression of alternatively spliced TnTf mRNAs in quail embryonic muscles. We report the following: 1) the quail TnTf gene is significantly larger than the rat TnTf gene and has 8 non-homologous exons, including a pectoral muscle-specific set of alternatively spliced exons; 2) specific sequences are implicated in regulated exon splicing; 3) a 900-base pair sequence element, composed primarily of intron sequence flanking the pectoral muscle-specific exons, is tandemly repeated 4 times and once partially, providing direct evidence that the pectoral-specific TnT exon domain arose by intragenic duplications; 4) a chicken repeat 1 retrotransposon element resides upstream of this repeated intronic/pectoral exon sequence domain and is implicated in transposition of this element into an ancestral genome; and 5) a large set of novel isoforms, produced by regulated exon splicing, is expressed in quail muscles, providing insights into the developmental regulation, physiological function, and evolution of the vertebrate TnTf isoforms.     

Genomic sequence and structural organization of mouse slow skeletal muscle troponin T gene

Three muscle type-specific troponin T (TnT) genes are present in vertebrate to encode a number of protein isoforms via alternative mRNA splicing. While the genomic structures of cardiac and fast skeletal muscle TnT genes have been documented, this study cloned and characterized the slow skeletal muscle TnT (sTnT) gene. Complete nucleotide sequence and genomic organization revealed that the mouse sTnT gene spans 11.1kb and contains 14 exons, which is smaller and simpler than the fast skeletal muscle and cardiac TnT genes. Potentially representing a prototype of the TnT gene family, the 5'-region of the sTnT gene contains fewer unsplit large exons, among which two alternatively spliced exons are responsible for the NH2-terminal variation of three sTnT isoforms. The sTnT gene structure shows that the alternatively spliced central segment found in human sTnT cDNAs may be a result from splicing using an alternative acceptor site at the intron 11-exon 12 boundary. Together with the well-conserved protein structure, the highly specific expression of sTnT in slow skeletal muscles indicates a differentiated function of this member of the TnT gene family. The determination of genomic structure and alternative splicing pathways of sTnT gene lays a foundation to further understand the TnT structure-function evolution as well as contractile characteristics of different types of muscle fiber.     

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.     

N-terminal amino acid sequences of three functionally different troponin T isoforms from rabbit fast skeletal muscle

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5' exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT1f, TnT2f and TnT3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT2f is the fast TnT sequenced by Pearlstone et al. The larger species TnT1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.     

Role of the fetal and alpha/beta exons in the function of fast skeletal troponin T isoforms: correlation with altered Ca2+ regulation associated with development

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, alpha (exon 16) and beta (exon 17), located near the 3' end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTalpha and the fetal TnTbeta isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5' end of the gene, is specifically present in TnTbeta and absent in the adult isoforms. To define the functional role of the f and alpha/beta exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTbeta cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTalpha, TnTalpha + f, TnTbeta - f and TnTbeta are identical, except for the presence or absence of the alpha or beta and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the alpha and beta peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.     

Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M_r to low M_r, acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.     

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.     

鸡形目鸟类成熟胸肌中特异性表达的fTnT同工异构型及其生理学意义

脊椎动物中的肌钙蛋白T（tropnin T,TnT）分为心肌型TnT（cardiac TnT,cTnT）、快肌型TnT（fast skeletal TnT,fTnT）和慢肌型TnT（slow skeletal TnT,sTnT）,且每种TnT又因mRNA可变剪接(alternative mRNA splicing)形成了多种同工异构型,其中fTnT的同工异构型形式最为复杂。某些鸟类如鸡形目鸟类的成熟快肌（尤其是胸部快肌）中特异性表达的TnT同工异构型有如下特点：（1）N端区含有过渡金属离子结合位点——Tx元件（一般为4~7个重复的H-E/A-E-A-H序列）;（2）与哺乳动物及雏鸟fTnT相比,其C端区外显子16有很高的表达率。本文还就鸡形目鸟类成熟胸肌中表达的fTnT同工异构型可能具有的生理学意义及应用前景进行了探讨。 The Fast TnT Isoforms Specifically Expressed in Avian Adult Pectoral Muscles of Galliforms and Physiological Significance DUAN Ying-li,YU Shu-yang,LI Ning National Laboratories for Agrobiotechnology,China Agricultural University,Beijing 100094,China Abstract:Three homologous genes have evolved to encode the cardiac,slow and fast skeletal muscle troponin Ts(TnTs) in the vertebrate.Multiple isoforms in each type of TnT are generated through alternative mRNA splicing during the development and the modality of the fast skeletal TnT isoforms is the most complex.The TnT isoforms specifically expressed in avian adult fast skeletal muscle (especially in the adult pectoral muscle) of Galliforms have been characterized as follows:1.There exist a cluster of transition metal ion binding sites ［generally 4~7 repeats of a sequence motif His-(Glu/Ala)- Glu-Ala-His,designated as Tx］ in the NH2-terminal variable region.2.Compared with mammalian TnT and the neonatal or young avian TnT,these avian pectoral muscle TnTs prefer to express exon 16 in the COOH-terminal variable region.Furthermore,possible effects of the pectoral fTnT isoforms on the physiological activity are discussed in this article. Key words：Aves; troponin T; isoform     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.