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Published on: October 28, 2014
Stabilization of slow troponin C polypeptide compensates for its reduced synthesis in antisense
1Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Abstract:
The expression of genes for contractile proteins during myogenesis is coordinately regulated. Uncoupling the expression of the slow/cardiac troponin C (sTnC) gene from this process with an antisense phosphorothioate oligodeoxynucleotide (ODN) was used to examine the presence of any post-transcriptional mechanisms for regulating muscle protein synthesis. Approximately 70 and 50% decreases in sTnC polypeptide synthesis and mRNA levels, respectively, were achieved after 4 days antisense treatment. This decrease in sTnC polypeptide synthesis was not reflected in a similar decline in the steady-state level of this polypeptide. Extension of the ODN treatment to 7 days was required to produce a substantial decrease in the steady-state level of sTnC polypeptide. Our investigation suggests that during the 4-day treatment, the affected cells stabilized the sTnC polypeptide level by increasing its half-life. However, the stabilizing effect appears to be overridden during prolonged (7 days) antisense ODN treatment. Measurement of the polypeptide synthesis and mRNA levels of several contractile proteins showed no evidence of cross-regulation among the genes to coordinately regulate their expression levels.
Insights
Antisense oligodeoxynucleotides (ODN) targeting slow/cardiac troponin C (sTnC) mRNA initially stabilized sTnC polypeptide levels. Prolonged treatment eventually reduced sTnC levels, indicating post-transcriptional regulation of muscle protein synthesis.
Area of Science:
- Molecular Biology
- Muscle Physiology
Background:
- Gene expression for contractile proteins is coordinately regulated during myogenesis.
- Post-transcriptional regulatory mechanisms for muscle protein synthesis are not fully understood.
Purpose of the Study:
- To investigate post-transcriptional regulation of muscle protein synthesis.
- To examine the effect of antisense oligodeoxynucleotides (ODN) on slow/cardiac troponin C (sTnC) gene expression and protein levels.
Main Methods:
- Utilized antisense phosphorothioate oligodeoxynucleotides (ODN) to inhibit sTnC gene expression.
- Measured sTnC polypeptide synthesis and mRNA levels over 4 and 7 days of treatment.
- Assessed steady-state levels of sTnC polypeptide.
Main Results:
- A 4-day antisense ODN treatment decreased sTnC polypeptide synthesis by 70% and mRNA by 50%.
- Steady-state sTnC polypeptide levels remained largely unchanged after 4 days, suggesting stabilization.
- A 7-day treatment was required for a substantial decrease in steady-state sTnC polypeptide levels.
- No evidence of cross-regulation was found among other contractile protein genes.
Conclusions:
- Muscle cells can stabilize sTnC polypeptide levels by increasing its half-life in response to short-term inhibition of gene expression.
- This stabilization effect is overcome with prolonged antisense ODN treatment.
- Coordinate regulation of contractile protein gene expression does not appear to involve cross-regulation at the post-transcriptional level studied.
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