Abstract
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.
Highlights
We describe the isolation and sequence analysis of quail muscle cDNA clones encodingtwo closely related isoformsof the striatedmuscle contractile protein, troponin T.The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship
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 d.(Medford, R
Because we identify cDNA clones corresponding to two different troponin T mRNAs (whereas only one of the two rat mRNAs has been identified by cloning [19]) our resultsconstitute importantnew evidence that directly demonstrates the occurrence of differential troponin T RNA splicing
Summary
We describe the isolation and sequence analysis of quail muscle cDNA clones encodingtwo closely related isoformsof the striatedmuscle contractile protein, troponin T.The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. 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 d.(Medford, R. Considering the complexity of these interactions, and thepossible importance of the specific molecular properties of the Ca2+switch in determining the characteristics of contractile activity, it is of great interest that troponin subunits occur in distinct isoformswhich are expressed in different muscle cell types. Six of these approximately 17mRNAspecieshavebeen identifiedas mRNAs encodingthe muscle contractile proteins a-actin, myosin heavy chain, myosin light chain 2, a-tropomyosin, troponin C, and troponin I [20,21].Among those cDNAs not identified in our previous studies we report
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