Alterations in the expression of muscle-specific genes mediated by troponin C antisense oligodeoxynucleotide.
The effectiveness of an antisense oligodeoxynucleotide to troponin C (TnC) mRNA in blocking expression of TnC in differentiated chicken myotubes was examined. An 18-nucleotide-long sequence common to both fast and slow isoforms of TnC mRNAs was chosen as the target sequence. The oligomer was found to be efficiently taken up by myotubes. However, the intracellular half-life of the oligomer was found to be only 3 h. Results of studies using different concentrations of oligomer for 3 h in the culture medium showed that compared to the untreated control culture, myotubes incubated with 20 microns antisense oligomer showed a 30% reduction in the steady-state level of TnC mRNAs. An increase of incubation period to 12 h with additions of fresh culture medium containing 20 microns antisense oligomer every 3 h failed to produce any further reduction of TnC mRNA level. Concomitant to the decrease of TnC mRNAs in antisense oligomer-treated cells, the steady-state levels of alpha-actin and alpha-tropomyosin mRNAs were also reduced by approximately 20 to 40%. Analysis of the homology of the sense sequence of this oligomer with that of alpha-actin and alpha-tropomyosin mRNAs suggested that reduction in the level of alpha-actin and alpha-tropomyosin mRNAs was not due to direct hybridization of the antisense oligomer to these mRNAs. Comparison of TnC polypeptide synthesis in untreated and oligomer-treated myotubes showed approximately 70% reduction of fast TnC polypeptide synthesis in antisense oligomer-treated cells. In contrast, slow TnC polypeptide synthesis was not significantly reduced in treated cells. Similarly, alpha-actin and alpha-tropomyosin polypeptide synthesis remained close to the level of untreated cells. Furthermore, analysis of transcription of various muscle-specific mRNAs showed increased synthesis of both TnC and alpha-tropomyosin mRNAs in antisense oligomer-treated myotubes. On the other hand, synthesis of actin mRNAs was not altered by this treatment. These results showed that antisense oligomer was effective in significantly reducing TnC polypeptide synthesis in chicken myotubes. Furthermore, these results suggest that treatment of myotubes with antisense oligomer to TnC mRNA may have triggered a complex array of compensatory processes.
Thinakaran
G
G
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
Bag
J
J
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics; D002642:Chick Embryo; D005786:Gene Expression Regulation / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D014335:Tropomyosin / Q000235:genetics; D014336:Troponin / Q000096:biosynthesis / Q000235:genetics*; D019209:Troponin C
D000199:Actins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014335:Tropomyosin; D014336:Troponin; D019209:Troponin C