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. 1996 Aug 2;134(4):827–835. doi: 10.1083/jcb.134.4.827

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region

PMCID: PMC2120959  PMID: 8769409

Abstract

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111- 1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.

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Selected References

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  1. Abastado J. P., Miller P. F., Hinnebusch A. G. A quantitative model for translational control of the GCN4 gene of Saccharomyces cerevisiae. New Biol. 1991 May;3(5):511–524. [PubMed] [Google Scholar]
  2. Arrick B. A., Lee A. L., Grendell R. L., Derynck R. Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region. Mol Cell Biol. 1991 Sep;11(9):4306–4313. doi: 10.1128/mcb.11.9.4306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atwater J. A., Wisdom R., Verma I. M. Regulated mRNA stability. Annu Rev Genet. 1990;24:519–541. doi: 10.1146/annurev.ge.24.120190.002511. [DOI] [PubMed] [Google Scholar]
  4. Degnin C. R., Schleiss M. R., Cao J., Geballe A. P. Translational inhibition mediated by a short upstream open reading frame in the human cytomegalovirus gpUL4 (gp48) transcript. J Virol. 1993 Sep;67(9):5514–5521. doi: 10.1128/jvi.67.9.5514-5521.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donzé O., Spahr P. F. Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging. EMBO J. 1992 Oct;11(10):3747–3757. doi: 10.1002/j.1460-2075.1992.tb05460.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dyson E., Sucov H. M., Kubalak S. W., Schmid-Schönbein G. W., DeLano F. A., Evans R. M., Ross J., Jr, Chien K. R. Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha -/- mice. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7386–7390. doi: 10.1073/pnas.92.16.7386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eisel U., Reynolds K., Riddick M., Zimmer A., Niemann H., Zimmer A. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis. EMBO J. 1993 Sep;12(9):3365–3372. doi: 10.1002/j.1460-2075.1993.tb06010.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geballe A. P., Morris D. R. Initiation codons within 5'-leaders of mRNAs as regulators of translation. Trends Biochem Sci. 1994 Apr;19(4):159–164. doi: 10.1016/0968-0004(94)90277-1. [DOI] [PubMed] [Google Scholar]
  10. Grant C. M., Miller P. F., Hinnebusch A. G. Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron. Mol Cell Biol. 1994 Apr;14(4):2616–2628. doi: 10.1128/mcb.14.4.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hart R. C., Winn K. J., Unger E. R. Avian model for 13-cis-retinoic acid embryopathy: morphological characterization of ventricular septal defects. Teratology. 1992 Dec;46(6):533–539. doi: 10.1002/tera.1420460602. [DOI] [PubMed] [Google Scholar]
  12. Hill J. R., Morris D. R. Cell-specific translation of S-adenosylmethionine decarboxylase mRNA. Regulation by the 5' transcript leader. J Biol Chem. 1992 Oct 25;267(30):21886–21893. [PubMed] [Google Scholar]
  13. Hill J. R., Morris D. R. Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Dependence on translation and coding capacity of the cis-acting upstream open reading frame. J Biol Chem. 1993 Jan 5;268(1):726–731. [PubMed] [Google Scholar]
  14. Kastner P., Grondona J. M., Mark M., Gansmuller A., LeMeur M., Decimo D., Vonesch J. L., Dollé P., Chambon P. Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell. 1994 Sep 23;78(6):987–1003. doi: 10.1016/0092-8674(94)90274-7. [DOI] [PubMed] [Google Scholar]
  15. Kessel M., Gruss P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell. 1991 Oct 4;67(1):89–104. doi: 10.1016/0092-8674(91)90574-i. [DOI] [PubMed] [Google Scholar]
  16. Kessel M., Gruss P. Open reading frames and translational control. Nature. 1988 Mar 10;332(6160):117–118. doi: 10.1038/332117c0. [DOI] [PubMed] [Google Scholar]
  17. Kessel M. Respecification of vertebral identities by retinoic acid. Development. 1992 Jun;115(2):487–501. doi: 10.1242/dev.115.2.487. [DOI] [PubMed] [Google Scholar]
  18. Kochhar D. M. Teratogenic activity of retinoic acid. Acta Pathol Microbiol Scand. 1967;70(3):398–404. doi: 10.1111/j.1699-0463.1967.tb01308.x. [DOI] [PubMed] [Google Scholar]
  19. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Res. 1984 May 11;12(9):3873–3893. doi: 10.1093/nar/12.9.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
  25. 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]
  26. Mendelsohn C., Lohnes D., Décimo D., Lufkin T., LeMeur M., Chambon P., Mark M. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development. 1994 Oct;120(10):2749–2771. doi: 10.1242/dev.120.10.2749. [DOI] [PubMed] [Google Scholar]
  27. Miller P. F., Hinnebusch A. G. Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control. Genes Dev. 1989 Aug;3(8):1217–1225. doi: 10.1101/gad.3.8.1217. [DOI] [PubMed] [Google Scholar]
  28. Molkentin J. D., Kalvakolanu D. V., Markham B. E. Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene. Mol Cell Biol. 1994 Jul;14(7):4947–4957. doi: 10.1128/mcb.14.7.4947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  30. Peabody D. S., Berg P. Termination-reinitiation occurs in the translation of mammalian cell mRNAs. Mol Cell Biol. 1986 Jul;6(7):2695–2703. doi: 10.1128/mcb.6.7.2695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  32. Reynolds K., Mezey E., Zimmer A. Activity of the beta-retinoic acid receptor promoter in transgenic mice. Mech Dev. 1991 Dec;36(1-2):15–29. doi: 10.1016/0925-4773(91)90068-h. [DOI] [PubMed] [Google Scholar]
  33. Schleiss M. R., Degnin C. R., Geballe A. P. Translational control of human cytomegalovirus gp48 expression. J Virol. 1991 Dec;65(12):6782–6789. doi: 10.1128/jvi.65.12.6782-6789.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sucov H. M., Dyson E., Gumeringer C. L., Price J., Chien K. R., Evans R. M. RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev. 1994 May 1;8(9):1007–1018. doi: 10.1101/gad.8.9.1007. [DOI] [PubMed] [Google Scholar]
  35. Twal W., Roze L., Zile M. H. Anti-retinoic acid monoclonal antibody localizes all-trans-retinoic acid in target cells and blocks normal development in early quail embryo. Dev Biol. 1995 Apr;168(2):225–234. doi: 10.1006/dbio.1995.1075. [DOI] [PubMed] [Google Scholar]
  36. Werner M., Feller A., Messenguy F., Piérard A. The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell. 1987 Jun 19;49(6):805–813. doi: 10.1016/0092-8674(87)90618-0. [DOI] [PubMed] [Google Scholar]
  37. Wiens D. J., Mann T. K., Fedderson D. E., Rathmell W. K., Franck B. H. Early heart development in the chick embryo: effects of isotretinoin on cell proliferation, alpha-actin synthesis, and development of contractions. Differentiation. 1992 Oct;51(2):105–112. doi: 10.1111/j.1432-0436.1992.tb00686.x. [DOI] [PubMed] [Google Scholar]
  38. Williams N. P., Mueller P. P., Hinnebusch A. G. The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences. Mol Cell Biol. 1988 Sep;8(9):3827–3836. doi: 10.1128/mcb.8.9.3827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yutzey K. E., Rhee J. T., Bader D. Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart. Development. 1994 Apr;120(4):871–883. doi: 10.1242/dev.120.4.871. [DOI] [PubMed] [Google Scholar]
  40. Zelent A., Mendelsohn C., Kastner P., Krust A., Garnier J. M., Ruffenach F., Leroy P., Chambon P. Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing. EMBO J. 1991 Jan;10(1):71–81. doi: 10.1002/j.1460-2075.1991.tb07922.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhou M. D., Sucov H. M., Evans R. M., Chien K. R. Retinoid-dependent pathways suppress myocardial cell hypertrophy. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7391–7395. doi: 10.1073/pnas.92.16.7391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zimmer A., Zimmer A. M., Reynolds K. Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region. J Cell Biol. 1994 Nov;127(4):1111–1119. doi: 10.1083/jcb.127.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zimmer A., Zimmer A. Induction of a RAR beta 2-lacZ transgene by retinoic acid reflects the neuromeric organization of the central nervous system. Development. 1992 Dec;116(4):977–983. doi: 10.1242/dev.116.4.977. [DOI] [PubMed] [Google Scholar]
  44. Zuker M. Computer prediction of RNA structure. Methods Enzymol. 1989;180:262–288. doi: 10.1016/0076-6879(89)80106-5. [DOI] [PubMed] [Google Scholar]

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