Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Oct 1;88(19):8606–8610. doi: 10.1073/pnas.88.19.8606

A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.

M L Gaspar 1, T Meo 1, P Bourgarel 1, J L Guenet 1, M Tosi 1
PMCID: PMC52558  PMID: 1924321

Abstract

Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation.

Full text

PDF
8606

Images in this article

8607Image
on p.8607
8607Image
on p.8607
8608Image
on p.8608
8608Image
on p.8608
8608Image
on p.8608
8609Image
on p.8609
8610Image
on p.8610
8610Image
on p.8610

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Attardi B., Ono S. Cytosol androgen receptor from kidney of normal and testicular feminized (Tfm) mice. Cell. 1974 Aug;2(4):205–212. doi: 10.1016/0092-8674(74)90012-9. [DOI] [PubMed] [Google Scholar]
  2. Atweh G. F., Brickner H. E., Zhu X. X., Kazazian H. H., Jr, Forget B. G. New amber mutation in a beta-thalassemic gene with nonmeasurable levels of mutant messenger RNA in vivo. J Clin Invest. 1988 Aug;82(2):557–561. doi: 10.1172/JCI113632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bardin C. W., Catterall J. F. Testosterone: a major determinant of extragenital sexual dimorphism. Science. 1981 Mar 20;211(4488):1285–1294. doi: 10.1126/science.7010603. [DOI] [PubMed] [Google Scholar]
  4. Baserga S. J., Benz E. J., Jr Nonsense mutations in the human beta-globin gene affect mRNA metabolism. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2056–2060. doi: 10.1073/pnas.85.7.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baumann B., Potash M. J., Köhler G. Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. EMBO J. 1985 Feb;4(2):351–359. doi: 10.1002/j.1460-2075.1985.tb03636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown T. R., Lubahn D. B., Wilson E. M., Joseph D. R., French F. S., Migeon C. J. Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8151–8155. doi: 10.1073/pnas.85.21.8151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bullock L. P., Bardin C. W. Androgen receptors in mouse kidney: a study of male, female and androgen-insensitive (tfm-y) mice. Endocrinology. 1974 Mar;94(3):746–756. doi: 10.1210/endo-94-3-746. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Daar I. O., Maquat L. E. Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol Cell Biol. 1988 Feb;8(2):802–813. doi: 10.1128/mcb.8.2.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dofuku R., Tettenborn U., Ohno S. Testosterone-"regulon" in the mouse kidney. Nat New Biol. 1971 Jul 7;232(27):5–7. doi: 10.1038/newbio232005a0. [DOI] [PubMed] [Google Scholar]
  11. Faber P. W., Kuiper G. G., van Rooij H. C., van der Korput J. A., Brinkmann A. O., Trapman J. The N-terminal domain of the human androgen receptor is encoded by one, large exon. Mol Cell Endocrinol. 1989 Feb;61(2):257–262. doi: 10.1016/0303-7207(89)90137-8. [DOI] [PubMed] [Google Scholar]
  12. Fox T. O., Wieland S. J. Isoelectric focusing of androgen receptors from wild-type and Tfm mouse kidneys. Endocrinology. 1981 Sep;109(3):790–797. doi: 10.1210/endo-109-3-790. [DOI] [PubMed] [Google Scholar]
  13. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  14. Gaspar M. L., Meo T., Tosi M. Structure and size distribution of the androgen receptor mRNA in wild-type and Tfm/Y mutant mice. Mol Endocrinol. 1990 Oct;4(10):1600–1610. doi: 10.1210/mend-4-10-1600. [DOI] [PubMed] [Google Scholar]
  15. Gehring U., Tomkins G. M. Characterization of a hormone receptor defect in the androgen-insensitivity mutant. Cell. 1974 Sep;3(1):59–64. doi: 10.1016/0092-8674(74)90040-3. [DOI] [PubMed] [Google Scholar]
  16. Herman R. C. Alternatives for the initiation of translation. Trends Biochem Sci. 1989 Jun;14(6):219–222. doi: 10.1016/0968-0004(89)90030-3. [DOI] [PubMed] [Google Scholar]
  17. Kadowaki T., Kadowaki H., Taylor S. I. A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(2):658–662. doi: 10.1073/pnas.87.2.658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kozak M. Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems. Mol Cell Biol. 1989 Nov;9(11):5073–5080. doi: 10.1128/mcb.9.11.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kozak M. Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 1990 May 11;18(9):2828–2828. doi: 10.1093/nar/18.9.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lipson K. E., Baserga R. Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9774–9777. doi: 10.1073/pnas.86.24.9774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lubahn D. B., Brown T. R., Simental J. A., Higgs H. N., Migeon C. J., Wilson E. M., French F. S. Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9534–9538. doi: 10.1073/pnas.86.23.9534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lubahn D. B., Joseph D. R., Sullivan P. M., Willard H. F., French F. S., Wilson E. M. Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science. 1988 Apr 15;240(4850):327–330. doi: 10.1126/science.3353727. [DOI] [PubMed] [Google Scholar]
  26. Lyon M. F., Hawkes S. G. X-linked gene for testicular feminization in the mouse. Nature. 1970 Sep 19;227(5264):1217–1219. doi: 10.1038/2271217a0. [DOI] [PubMed] [Google Scholar]
  27. Marcelli M., Tilley W. D., Wilson C. M., Griffin J. E., Wilson J. D., McPhaul M. J. Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance. Mol Endocrinol. 1990 Aug;4(8):1105–1116. doi: 10.1210/mend-4-8-1105. [DOI] [PubMed] [Google Scholar]
  28. Marcelli M., Tilley W. D., Wilson C. M., Wilson J. D., Griffin J. E., McPhaul M. J. A single nucleotide substitution introduces a premature termination codon into the androgen receptor gene of a patient with receptor-negative androgen resistance. J Clin Invest. 1990 May;85(5):1522–1528. doi: 10.1172/JCI114599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ono S., Geller L. N., Lai E. V. TfM mutation and masculinization versus feminization of the mouse central nervous system. Cell. 1974 Nov;3(3):235–242. doi: 10.1016/0092-8674(74)90137-8. [DOI] [PubMed] [Google Scholar]
  30. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  31. Politch J. A., Fox T. O., Houben P., Bullock L., Lovell D. TfmLac: a second isolation of testicular feminization in mice. Biochem Genet. 1988 Apr;26(3-4):213–221. doi: 10.1007/BF00561461. [DOI] [PubMed] [Google Scholar]
  32. Prats H., Kaghad M., Prats A. C., Klagsbrun M., Lélias J. M., Liauzun P., Chalon P., Tauber J. P., Amalric F., Smith J. A. High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1836–1840. doi: 10.1073/pnas.86.6.1836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Quarmby V. E., Yarbrough W. G., Lubahn D. B., French F. S., Wilson E. M. Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol. 1990 Jan;4(1):22–28. doi: 10.1210/mend-4-1-22. [DOI] [PubMed] [Google Scholar]
  34. Ris-Stalpers C., Kuiper G. G., Faber P. W., Schweikert H. U., van Rooij H. C., Zegers N. D., Hodgins M. B., Degenhart H. J., Trapman J., Brinkmann A. O. Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7866–7870. doi: 10.1073/pnas.87.20.7866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sai T. J., Seino S., Chang C. S., Trifiro M., Pinsky L., Mhatre A., Kaufman M., Lambert B., Trapman J., Brinkmann A. O. An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity. Am J Hum Genet. 1990 Jun;46(6):1095–1100. [PMC free article] [PubMed] [Google Scholar]
  36. Schenkein I., Levy M., Bueker E. D., Wilson J. D. Immunological and enzymatic evidence for the absence of an esteroproteolytic enzyme (protease "D") in the submandibular gland of the Tfm mouse. Endocrinology. 1974 Mar;94(3):840–844. doi: 10.1210/endo-94-3-840. [DOI] [PubMed] [Google Scholar]
  37. Toole J. J., Hastie N. D., Held W. A. An abundant androgen-regulated mRNA in the mouse kidney. Cell. 1979 Jun;17(2):441–448. doi: 10.1016/0092-8674(79)90170-3. [DOI] [PubMed] [Google Scholar]
  38. Young C. Y., Johnson M. P., Prescott J. L., Tindall D. J. The androgen receptor of the testicular-feminized (Tfm) mutant mouse is smaller than the wild-type receptor. Endocrinology. 1989 Feb;124(2):771–775. doi: 10.1210/endo-124-2-771. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES