Abstract
The human dystrophin gene has 79 exons spanning >2300 kb making it the largest known gene. In previous studies we showed that approximately 16 h are required to transcribe the gene in myogenic cultures [Tennyson, C.N., Klamut, H.J. and Worton, R.G. (1995) Nature Genet. 9, 184-190]. To estimate the half-life of the dystrophin mRNA, the decay of the transcript was monitored by quantitative RT-PCR in cultured human fetal myotubes following exposure to actinomycin D. Results from this analysis indicated that the half-life of the dystrophin mRNA is 15.6 +/- 2.8 h in these cultures. Transcript accumulation profiles were predicted using a mathematical model which incorporated the measured half-life. The modeled accumulation profiles were consistent with observed profiles supporting the half-life measured using actinomycin D. The kinetic model was then used to predict the relative amount of nascent and mature dystrophin transcript at steady state. Measurements by quantitative RT-PCR indicated that in adult skeletal muscle tissue the concentration of mature dystrophin mRNA is 5-10 molecules per nucleus, demonstrating, as expected, that it is a low abundance transcript. Furthermore the ratio of nascent to mature dystrophin transcript indicated that dystrophin synthesis may not be at steady state in the adult skeletal muscle we tested. Alternatively, the kinetics of transcript production in skeletal muscle tissue may be different from those observed in cultured fetal myogenic cells.
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- Bies R. D., Phelps S. F., Cortez M. D., Roberts R., Caskey C. T., Chamberlain J. S. Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development. Nucleic Acids Res. 1992 Apr 11;20(7):1725–1731. doi: 10.1093/nar/20.7.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campbell K. P., Kahl S. D. Association of dystrophin and an integral membrane glycoprotein. Nature. 1989 Mar 16;338(6212):259–262. doi: 10.1038/338259a0. [DOI] [PubMed] [Google Scholar]
- Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [DOI] [PubMed] [Google Scholar]
- Chelly J., Montarras D., Pinset C., Berwald-Netter Y., Kaplan J. C., Kahn A. Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells. Eur J Biochem. 1990 Feb 14;187(3):691–698. doi: 10.1111/j.1432-1033.1990.tb15355.x. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Coffey A. J., Roberts R. G., Green E. D., Cole C. G., Butler R., Anand R., Giannelli F., Bentley D. R. Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach. Genomics. 1992 Mar;12(3):474–484. doi: 10.1016/0888-7543(92)90437-w. [DOI] [PubMed] [Google Scholar]
- Feener C. A., Koenig M., Kunkel L. M. Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature. 1989 Apr 6;338(6215):509–511. doi: 10.1038/338509a0. [DOI] [PubMed] [Google Scholar]
- Gibson M. C., Schultz E. Age-related differences in absolute numbers of skeletal muscle satellite cells. Muscle Nerve. 1983 Oct;6(8):574–580. doi: 10.1002/mus.880060807. [DOI] [PubMed] [Google Scholar]
- Hahn C. G., Covault J. Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients. Anal Biochem. 1990 Nov 1;190(2):193–197. doi: 10.1016/0003-2697(90)90180-h. [DOI] [PubMed] [Google Scholar]
- Hargrove J. L., Schmidt F. H. The role of mRNA and protein stability in gene expression. FASEB J. 1989 Oct;3(12):2360–2370. doi: 10.1096/fasebj.3.12.2676679. [DOI] [PubMed] [Google Scholar]
- Klamut H. J., Zubrzycka-Gaarn E. E., Bulman D. E., Malhotra S. B., Bodrug S. E., Worton R. G., Ray P. N. Myogenic regulation of dystrophin gene expression. Br Med Bull. 1989 Jul;45(3):681–702. doi: 10.1093/oxfordjournals.bmb.a072352. [DOI] [PubMed] [Google Scholar]
- Koenig M., Monaco A. P., Kunkel L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 1988 Apr 22;53(2):219–228. doi: 10.1016/0092-8674(88)90383-2. [DOI] [PubMed] [Google Scholar]
- Lederfein D., Yaffe D., Nudel U. A housekeeping type promoter, located in the 3' region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene. Hum Mol Genet. 1993 Nov;2(11):1883–1888. doi: 10.1093/hmg/2.11.1883. [DOI] [PubMed] [Google Scholar]
- Medford R. M., Nguyen H. T., Nadal-Ginard B. Transcriptional and cell cycle-mediated regulation of myosin heavy chain gene expression during muscle cell differentiation. J Biol Chem. 1983 Sep 25;258(18):11063–11073. [PubMed] [Google Scholar]
- Monaco A. P., Walker A. P., Millwood I., Larin Z., Lehrach H. A yeast artificial chromosome contig containing the complete Duchenne muscular dystrophy gene. Genomics. 1992 Mar;12(3):465–473. doi: 10.1016/0888-7543(92)90436-v. [DOI] [PubMed] [Google Scholar]
- Nevins J. R., Darnell J. E., Jr Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell. 1978 Dec;15(4):1477–1493. doi: 10.1016/0092-8674(78)90071-5. [DOI] [PubMed] [Google Scholar]
- Nudel U., Robzyk K., Yaffe D. Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain. Nature. 1988 Feb 18;331(6157):635–638. doi: 10.1038/331635a0. [DOI] [PubMed] [Google Scholar]
- Roberts R. G., Coffey A. J., Bobrow M., Bentley D. R. Exon structure of the human dystrophin gene. Genomics. 1993 May;16(2):536–538. doi: 10.1006/geno.1993.1225. [DOI] [PubMed] [Google Scholar]
- Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
- Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
- Tennyson C. N., Klamut H. J., Worton R. G. The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nat Genet. 1995 Feb;9(2):184–190. doi: 10.1038/ng0295-184. [DOI] [PubMed] [Google Scholar]
- Volloch V., Housman D. Stability of globin mRNA in terminally differentiating murine erythroleukemia cells. Cell. 1981 Feb;23(2):509–514. doi: 10.1016/0092-8674(81)90146-x. [DOI] [PubMed] [Google Scholar]
- Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson T., Treisman R. Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature. 1988 Nov 24;336(6197):396–399. doi: 10.1038/336396a0. [DOI] [PubMed] [Google Scholar]
- Wisdom R., Lee W. Translation of c-myc mRNA is required for its post-transcriptional regulation during myogenesis. J Biol Chem. 1990 Nov 5;265(31):19015–19021. [PubMed] [Google Scholar]
- Zubrzycka-Gaarn E. E., Bulman D. E., Karpati G., Burghes A. H., Belfall B., Klamut H. J., Talbot J., Hodges R. S., Ray P. N., Worton R. G. The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature. 1988 Jun 2;333(6172):466–469. doi: 10.1038/333466a0. [DOI] [PubMed] [Google Scholar]