Abstract
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.
Highlights
Show that the N-terminal region of troponin T binds stably adult cardiac muscles or in non-muscle tissues
The alternative exon sequences TnTf mRNAs splicing of asinglegene in the N- and C-terminal regions expressed in anatomically distinct quail muscles can [15,16,17]
Clonal Analysis of a and p Expression in Myogenic Cultures-We examined whether thceoexpression of a and p in embryonic muscle reflects a heterogeneity of myogenic lineages, each of which express only one splice form, a or 0
Summary
XAR muscles expressrestrictedcombinations of splicechoices, indicating that TnTf isoform expression is regulated at the RESULTS. We screened these libraries witha partial sequences encoded by regulated, alternatively spliced exons quail T n T cDNA, cC122, and with oligonucleotides complein both bird and mammgaelnes are divergent,suggesting that mentary to alternative exonsencoding amino acids 229-242, these domains are important for specialized muscle function. CDNA Cloning and Screening-We generated cDNA libraries from 5 gg of poly(A+) mRNA isolated from cultured pectoral myofibers myofiber cDNAs identified seven positivephage, two of which were a and five of which were 0.One a clone, cC501, is nearly [21] and adult leg muscle. Isolation and Sequencing of TnT Gen.omic Clone gC106-gC106 is a recombinant Charon 4A phage that was isolated by screening an EcoRI partial embryonic quail genomic DNA library in A Charon 4A using the rat cDNA clone pTnT-15 as a probe [18, 32].
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