Bucher E A, de la Brousse F C, Emerson C P
Department of Biology, University of Virginia, Charlottesville 22901.
J Biol Chem. 1989 Jul 25;264(21):12482-91.
We have investigated the developmental regulation of the avian fast skeletal muscle troponin T (TnTf) gene of the Japanese quail. Sequence analysis of troponin T mRNA, cDNA clones, and a genomic DNA segment demonstrate that the avian, fast skeletal TnTf protein isoforms are produced from a single gene. This TnTf gene is expressed in skeletal muscle, but not in adult cardiac muscles or in non-muscle tissues. In addition to known TnT isoforms, three new isoforms of TnT are described. These isoforms arise by regulated alternative RNA splicing of exons in the 5' and 3' regions of TnTf transcripts. Alternative splicing of the 5' TnTf exons involves splicing of multiple exons in different combinations (i.e. not mutually exclusive), whereas 3' alternative splicing involves mutually exclusive splice choices between two exons (alpha or beta exons). S1 nuclease protection and primer extension analyses show that alternative splicing of both 5' and 3' exons is precisely regulated and coordinated in physiologically different striated muscles, which express distinct, restricted combinations of 5' and 3' alternatively spliced exons in mRNA transcripts. In contrast, different embryonic muscles and clonal embryonic myoblast cultures coexpress the 3' alternative splice choices. This indicates that alternative splicing of TnTf mRNAs is controlled in different adult muscles by specific trans factors, and not by the restricted expression of different spliced forms in different embryonic myoblast lineages. Comparison of TnTf isoform expression in quail and chicken flight muscle (Wilkinson, J. M., Moir, A. J., and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47-56) to TnTf isoforms of the rat (Breitbart, R. E., and Nadal-Ginard, B. (1986) J. Mol. Biol. 188, 313-324), and rabbit (Pearlstone, J. R., Carpenter, M. R., and Smillie, M. B. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1902-1906) indicates that the avian gene contains an additional exon(s) not present in mammalian genes. The alternative exon sequences TnTf mRNAs expressed in anatomically distinct quail muscles can be correlated with sequences in TnTf protein isoforms in these chicken muscles. Thus, the regulated splicing of alternative exons in TnT transcripts, and not selective translation of stochastically spliced TnT mRNAs, regulates TnTf isoform expression in specific muscles.
我们研究了日本鹌鹑禽快速骨骼肌肌钙蛋白T（TnTf）基因的发育调控。对肌钙蛋白T mRNA、cDNA克隆和基因组DNA片段的序列分析表明，禽快速骨骼肌TnTf蛋白异构体由单个基因产生。该TnTf基因在骨骼肌中表达，但在成体心肌或非肌肉组织中不表达。除了已知的TnT异构体，还描述了三种新的TnT异构体。这些异构体通过TnTf转录本5'和3'区域外显子的可变RNA剪接产生。5'TnTf外显子的可变剪接涉及多个外显子以不同组合进行剪接（即不是相互排斥的），而3'可变剪接涉及两个外显子（α或β外显子）之间相互排斥的剪接选择。S1核酸酶保护和引物延伸分析表明，5'和3'外显子的可变剪接在生理上不同的横纹肌中受到精确调控和协调，这些横纹肌在mRNA转录本中表达5'和3'可变剪接外显子的不同、受限组合。相反，不同的胚胎肌肉和克隆胚胎成肌细胞培养物共同表达3'可变剪接选择。这表明TnTf mRNA的可变剪接在不同的成体肌肉中由特定的反式因子控制，而不是由不同胚胎成肌细胞谱系中不同剪接形式的受限表达控制。将鹌鹑和鸡飞行肌中TnTf异构体的表达（Wilkinson, J. M., Moir, A. J., and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47 - 56）与大鼠（Breitbart, R. E., and Nadal - Ginard, B. (1986) J. Mol. Biol. 188, 313 - 324）和兔子（Pearlstone, J. R., Carpenter, M. R., and Smillie, M. B. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1902 - 1906）的TnTf异构体进行比较，表明禽基因包含一个哺乳动物基因中不存在的额外外显子。在解剖学上不同的鹌鹑肌肉中表达的TnTf mRNA的可变外显子序列可以与这些鸡肌肉中TnTf蛋白异构体的序列相关联。因此，TnT转录本中可变外显子的调控剪接，而不是随机剪接的TnT mRNA的选择性翻译，调节特定肌肉中TnTf异构体的表达。
