Muscle-Specific Splicing of a Heterologous Exon Mediated by a Single Muscle-Specific Splicing Enhancer from the Cardiac Troponin T Gene

ABSTRACT The chicken cardiac troponin T (cTNT) gene contains a single 30-nucleotide alternative exon that is included in embryonic striated muscle and skipped in the adult. Transient-transfection analysis of cTNT minigenes in muscle and fibroblast cell cultures previously identified four muscle-specific splicing enhancers (MSEs) that promote exon inclusion specifically in embryonic striated muscle cultures. Three MSEs located in the intron downstream from the alternative exon were sufficient for muscle-specific exon inclusion. In the present study, the boundaries of these MSEs were defined by scanning mutagenesis, allowing analysis of individual elements in gain-of-function experiments. Concatamers of MSE2 were necessary and sufficient to promote muscle-specific inclusion of a heterologous exon, indicating that it is a target for muscle-specific regulation. Sequences present in MSE2 are also found in MSE4, suggesting that these two MSEs act in a similar manner. MSE3 appears to be different from MSE2 and MSE4 yet is able to functionally replace both of these elements, demonstrating functional redundancy of elements that are likely to bind different factors. MSE2 and MSE4 each contain a novel sequence motif that is found adjacent to a number of alternative exons that undergo regulated splicing in striated muscle, suggesting a common role for this element in muscle-specific regulation.

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A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4898 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4898-4907 ◽

Cited By ~ 92

Author(s):

J Ramchatesingh ◽

A M Zahler ◽

K M Neugebauer ◽

M B Roth ◽

T A Cooper

Keyword(s):

Cardiac Troponin ◽

Troponin T ◽

Sr Proteins ◽

Alternative Exon ◽

Cross Linking ◽

Cardiac Troponin T ◽

Sr Protein ◽

Exon Inclusion ◽

Enhancer Rna

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.

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The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3660 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3660-3674 ◽

Cited By ~ 89

Author(s):

R Xu ◽

J Teng ◽

T A Cooper

Keyword(s):

Splice Site ◽

Cardiac Troponin ◽

Troponin T ◽

Alternative Exon ◽

Cardiac Troponin T ◽

Splice Sites ◽

Developmentally Regulated ◽

Sequence Comparisons ◽

Muscle Cultures

We have characterized a novel positive-acting splicing element within the developmentally regulated alternative exon (exon 5) of the cardiac troponin T (cTNT) gene. The exon splicing element (ESE) is internal to the exon portions of the splice sites and is required for splicing to the 3' splice site but not the 5' splice site flanking the exon. Sequence comparisons between cTNT exon 5 and other exons that contain regions required for splicing reveal a common purine-rich motif. Sequence within cTNT exon 5 or a synthetic purine-rich motif facilitates splicing of heterologous alternative and constitutive splice sites in vivo. Interestingly, the ESE is not required for the preferential inclusion of cTNT exon 5 observed in primary skeletal muscle cultures. Our results strongly suggest that the purine-rich ESE serves as a general splicing element that is recognized by the constitutive splicing machinery.

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The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3660-3674.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3660-3674

Author(s):

R Xu ◽

J Teng ◽

T A Cooper

Keyword(s):

Splice Site ◽

Cardiac Troponin ◽

Troponin T ◽

Alternative Exon ◽

Cardiac Troponin T ◽

Splice Sites ◽

Developmentally Regulated ◽

Sequence Comparisons ◽

Muscle Cultures

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Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4014 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4014-4023 ◽

Cited By ~ 57

Author(s):

K J Ryan ◽

T A Cooper

Keyword(s):

Skeletal Muscle ◽

Alternative Splicing ◽

Cardiac Troponin ◽

Troponin T ◽

Alternative Exon ◽

Specific Gene ◽

Cardiac Troponin T ◽

Related Sequence ◽

Exon Inclusion ◽

Splicing Pattern

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.

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Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing

Nucleic Acids Research ◽

10.1093/nar/17.19.7905 ◽

1989 ◽

Vol 17 (19) ◽

pp. 7905-7921 ◽

Cited By ~ 28

Author(s):

Thomas A. Cooper ◽

Charles P. Ordahl

Keyword(s):

Alternative Splicing ◽

Cardiac Troponin ◽

Troponin T ◽

Alternative Exon ◽

Cardiac Troponin T ◽

Nucleotide Substitutions

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Stepwise C-Terminal Truncation of Cardiac Troponin T Alters Function at Low and Saturating Ca2+

10.1101/304642 ◽

2018 ◽

Author(s):

D. Johnson ◽

W. Angus ◽

J.M. Chalovich

Keyword(s):

Cardiac Troponin ◽

Striated Muscle ◽

Atp Hydrolysis ◽

Troponin T ◽

Troponin C ◽

Cardiac Troponin T ◽

C Terminus ◽

Myosin S1 ◽

Stimulation Of

AbstractActivation of striated muscle contraction occurs in response to Ca2+ binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+ are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+ stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+ and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+ and for limiting the formation of the active M state at saturating Ca2+.

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