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

1995 ◽  
Vol 15 (9) ◽  
pp. 4898-4907 ◽  
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.

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

1998 ◽  
Vol 18 (8) ◽  
pp. 4519-4525 ◽  
Author(s):  
Thomas A. Cooper
Keyword(s):  
Cardiac Troponin ◽  
Striated Muscle ◽  
Troponin T ◽  
Fibroblast Cell ◽  
Alternative Exon ◽  
Sequence Motif ◽  
Cardiac Troponin T ◽  
Exon Inclusion ◽  
Muscle Cultures ◽  
Specific Regulation

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.

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

1996 ◽  
Vol 16 (8) ◽  
pp. 4014-4023 ◽  
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.

306 IN VITRO CARDIOMYOGENIC AND NEUROGENIC DIFFERENTIATION OF MINIPIG BONE MARROW MESENCHYMAL STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 249
Author(s):  
B. Mohana Kumar ◽  
T. H. Kim ◽  
Y. M. Lee ◽  
G. H. Maeng ◽  
B. G. Jeon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cardiac Troponin ◽  
Troponin T ◽  
Cardiac Troponin T ◽  
Rt Pcr ◽  
Neurogenic Differentiation ◽  
Cardiac Actin

Differentiation of mesenchymal stem cells (MSC) into specialised cells in vitro before transplantation may improve the engraftment efficiency of the transplanted cells as well as the safety and efficacy of treatment. To understand the differentiation process and the functional identities of cells in an animal model, we examined the in vitro differentiation capacity of porcine MSC (3–6 passage) into cardiomyocyte-like and neuron-like cells. The MSC isolated from the bone marrow of postnatal miniature piglets [T-type, PWG Micro-pig (R), PWG Genetics, Korea] exhibited a typical fibroblast-like morphology and expressed the specific markers, such as CD29, CD44, and CD90. After 21 days of culture in induction media, MSC revealed the appropriate phenotype of osteocytes (von Kossa and Alizarin red), adipocytes (Oil red O), and chondrocytes (Alcian blue). Ther MSC were further induced into cardiomyogenic and neurogenic differentiation following the protocols described earlier (Tomita et al. 2002 J. Thorac. Cardiovasc. Surg. 123, 1132–1140) and (Woodbury et al. 2002 J. Neurosci. Res. 96, 908–917), respectively, with minor modifications. Expression of lineage-specific markers was evaluated by immunocytochemistry, and RT-PCR and quantitative PCR (RT-qPCR). For cardiomyogenic differentiation, MSC were stimulated with 10 μM 5-azacytidine for 24 h, 3 days, or 7 days, and the cells were maintained in culture for 21 days. Upon induction, MSC exhibited elongated and stick-like morphology with extended cytoplasmic processes, and toward the end of culture, cells formed aggregates and myotube-like structures. Immunostaining was positive for the markers of cardiomyocyte-like cells, such as α-smooth muscle actin, cardiac troponin T, desmin, and α-cardiac actin. The RT-PCR and RT-qPCR analysis showed the expression and a time dependent up-regulation of cardiac troponin T, desmin, α-cardiac actin, and β-myosin heavy chain genes. Following induction with neuronal-specific media for 3 days, above 80% of MSC acquired the morphology of neuron-like cells with bi- or multipolar cell processes forming a network-like structure. Induced cells with neuronal phenotype were positively stained for nestin, neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and neurofilament-M (NF-M). The expression of neural transcripts, such as nestin, GFAP, and NF-M, was further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the potential of porcine MSC to differentiate in vitro into cardiomyocyte-like and neuron-like cells, thus offering a useful model for studying their functional and molecular properties before transplantation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

Abstract 62: Cardiac Troponin T Isoform Switching in Early Childhood Tropomyosin-linked Dilated Cardiomyopathy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Melissa Lynn ◽  
Lauren Tal-Grinspan ◽  
J.-P. Jin ◽  
Jil Tardiff
Keyword(s):  
Mouse Model ◽  
Cardiac Troponin ◽  
Troponin T ◽  
Systolic Function ◽  
Phenotypic Variability ◽  
Bimodal Distribution ◽  
General Mechanism ◽  
Cardiac Troponin T ◽  
Human Heart Failure

An oft-noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. In a recent study of two unrelated multigenerational families with the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (<1 year) with the mutation presented with a severe form of DCM that led to sudden, often fatal CHF while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. A potential hypothesis to explain this improvement despite the continued presence of the mutant Tm, is that the phenotype is modified by other thin filament isoforms. Thus we propose that the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). Our initial biophysical studies (Regulated-IVM) revealed a decreased Ca2+ sensitivity in filaments containing D230N Tm that is more severe in the presence of fetal TnT (cTnT1), suggesting a modulatory role for cTnT1. Cardiac performance, assessed via 2D echo, in our novel D230N Tm x cTnT1 double transgenic (DTg) mouse model found a significantly reduced % FS for DTg (17%) mice as compared to D230N Tm (21%) littermates. This reduction in %FS was seen at 4 months but not 2 suggesting a progressive cardiomyopathy. Current efforts aim to model the early phase of this “bimodal” phenotype and assess the potential for disease reversibility using a cardiac specific inducible cTnT1 transgenic mouse model. Furthermore, we propose that modulation by cTnT1 could represent a more general mechanism for the progressive remodeling seen in human heart failure. Preliminary in vitro studies with human tissue found that RNA levels of cTnT1 are significantly higher in failing hearts as compared to non-failing. Thus these data suggest an isoform dependent mechanism for the “bimodal” phenotype in patients carrying D230N Tm that could translate to other sarcomeric cardiomyopathies.

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex

1994 ◽  
Vol 14 (11) ◽  
pp. 7670-7682
Author(s):  
D Staknis ◽  
R Reed
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Sr Proteins ◽  
Cross Linking ◽  
Splice Sites ◽  
Sr Protein ◽  
Specific Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

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

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

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.

Development and In Vitro Characterization of a New Immunoassay of Cardiac Troponin T

1992 ◽  
Vol 38 (3) ◽  
pp. 386-393 ◽  
Author(s):  
H A Katus ◽  
S Looser ◽  
K Hallermayer ◽  
A Remppis ◽  
T Scheffold ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cardiac Troponin ◽  
Solid Phase ◽  
Troponin T ◽  
Immunoblot Analysis ◽  
T Antigen ◽  
Cardiac Troponin T ◽  
In Vitro Characterization ◽  
Hybridoma Technology

Abstract We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificity of the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [Enzymun-TestR system (Boehringer Mannheim GmbH)] is performed in only 90 min at room temperature; the measuring range for troponin T is 0.1 to 15 micrograms/L. The assay shows excellent between-run precision (CV = 3.3-4.9%).

Sign in / Sign up

Export Citation Format

Share Document