Skip to main content
NCBI home page
RNA logoLink to RNA
. 2001 Dec;7(12):1753–1767.

A 250-nucleotide UA-rich element in the 3' untranslated region of Xenopus laevis Vg1 mRNA represses translation both in vivo and in vitro.

L J Otero 1, A Devaux 1, N Standart 1
PMCID: PMC1370215  PMID: 11780632

Abstract

Xenopus laevis Vgl mRNA undergoes both localization and translational control during oogenesis. Vg1 protein does not appear until late stage IV, after localization is complete. To determine whether Vg1 translation is regulated by cytoplasmic polyadenylation, the RACE-PAT method was used. Vg1 mRNA has a constant poly(A) tail throughout oogenesis, precluding a role for cytoplasmic polyadenylation. To identify cis-acting elements involved in Vg1 translational control, the Vg1 3' UTR was inserted downstream of the luciferase ORF and in vitro transcribed, adenylated mRNA injected into stage III or stage VI oocytes. The Vg1 3' UTR repressed luciferase translation in both stages. Deletion analysis of the Vg1 3' UTR revealed that a 250-nt UA-rich fragment, the Vg1 translational element or VTE, which lies 118 nt downstream of the Vg1 localization element, could repress translation as well as the full-length Vg1 3' UTR. Poly(A)-dependent translation is not necessary for repression as nonadenylated mRNAs are also repressed, but cap-dependent translation is required as introduction of the classical swine fever virus IRES upstream of the luciferase coding region prevents repression by the VTE. Repression by the Vg1 3' UTR has been reproduced in Xenopus oocyte in vitro translation extracts, which show a 10-25-fold synergy between the cap and poly(A) tail. A number of proteins UV crosslink to the VTE including FRGY2 and proteins of 36, 42, 45, and 60 kDa. The abundance of p42, p45, and p60 is strikingly higher in stages I-III than in later stages, consistent with a possible role for these proteins in Vg1 translational control.

Full Text

The Full Text of this article is available as a PDF (803.0 KB).

Selected References

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

  1. Asaoka-Taguchi M., Yamada M., Nakamura A., Hanyu K., Kobayashi S. Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos. Nat Cell Biol. 1999 Nov;1(7):431–437. doi: 10.1038/15666. [DOI] [PubMed] [Google Scholar]
  2. Barkoff A. F., Dickson K. S., Gray N. K., Wickens M. Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation. Dev Biol. 2000 Apr 1;220(1):97–109. doi: 10.1006/dbio.2000.9613. [DOI] [PubMed] [Google Scholar]
  3. Bashirullah A., Cooperstock R. L., Lipshitz H. D. RNA localization in development. Annu Rev Biochem. 1998;67:335–394. doi: 10.1146/annurev.biochem.67.1.335. [DOI] [PubMed] [Google Scholar]
  4. Bergamini G., Preiss T., Hentze M. W. Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA. 2000 Dec;6(12):1781–1790. doi: 10.1017/s1355838200001679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergsten S. E., Gavis E. R. Role for mRNA localization in translational activation but not spatial restriction of nanos RNA. Development. 1999 Feb;126(4):659–669. doi: 10.1242/dev.126.4.659. [DOI] [PubMed] [Google Scholar]
  6. Caput D., Beutler B., Hartog K., Thayer R., Brown-Shimer S., Cerami A. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1670–1674. doi: 10.1073/pnas.83.6.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castagnetti S., Hentze M. W., Ephrussi A., Gebauer F. Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries. Development. 2000 Mar;127(5):1063–1068. doi: 10.1242/dev.127.5.1063. [DOI] [PubMed] [Google Scholar]
  8. Clark I. E., Wyckoff D., Gavis E. R. Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism. Curr Biol. 2000 Oct 19;10(20):1311–1314. doi: 10.1016/s0960-9822(00)00754-5. [DOI] [PubMed] [Google Scholar]
  9. Cote C. A., Gautreau D., Denegre J. M., Kress T. L., Terry N. A., Mowry K. L. A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization. Mol Cell. 1999 Sep;4(3):431–437. doi: 10.1016/s1097-2765(00)80345-7. [DOI] [PubMed] [Google Scholar]
  10. Crucs S., Chatterjee S., Gavis E. R. Overlapping but distinct RNA elements control repression and activation of nanos translation. Mol Cell. 2000 Mar;5(3):457–467. doi: 10.1016/s1097-2765(00)80440-2. [DOI] [PubMed] [Google Scholar]
  11. Curtis D., Lehmann R., Zamore P. D. Translational regulation in development. Cell. 1995 Apr 21;81(2):171–178. doi: 10.1016/0092-8674(95)90325-9. [DOI] [PubMed] [Google Scholar]
  12. Dahanukar A., Walker J. A., Wharton R. P. Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila. Mol Cell. 1999 Aug;4(2):209–218. doi: 10.1016/s1097-2765(00)80368-8. [DOI] [PubMed] [Google Scholar]
  13. Dahanukar A., Wharton R. P. The Nanos gradient in Drosophila embryos is generated by translational regulation. Genes Dev. 1996 Oct 15;10(20):2610–2620. doi: 10.1101/gad.10.20.2610. [DOI] [PubMed] [Google Scholar]
  14. Dale L., Matthews G., Tabe L., Colman A. Developmental expression of the protein product of Vg1, a localized maternal mRNA in the frog Xenopus laevis. EMBO J. 1989 Apr;8(4):1057–1065. doi: 10.1002/j.1460-2075.1989.tb03473.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deshler J. O., Highett M. I., Abramson T., Schnapp B. J. A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates. Curr Biol. 1998 Apr 23;8(9):489–496. doi: 10.1016/s0960-9822(98)70200-3. [DOI] [PubMed] [Google Scholar]
  16. Ephrussi A., Dickinson L. K., Lehmann R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991 Jul 12;66(1):37–50. doi: 10.1016/0092-8674(91)90137-n. [DOI] [PubMed] [Google Scholar]
  17. Gautreau D., Cote C. A., Mowry K. L. Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes. Development. 1997 Dec;124(24):5013–5020. doi: 10.1242/dev.124.24.5013. [DOI] [PubMed] [Google Scholar]
  18. Gavis E. R., Lehmann R. Localization of nanos RNA controls embryonic polarity. Cell. 1992 Oct 16;71(2):301–313. doi: 10.1016/0092-8674(92)90358-j. [DOI] [PubMed] [Google Scholar]
  19. Gavis E. R., Lehmann R. Translational regulation of nanos by RNA localization. Nature. 1994 May 26;369(6478):315–318. doi: 10.1038/369315a0. [DOI] [PubMed] [Google Scholar]
  20. Gavis E. R., Lunsford L., Bergsten S. E., Lehmann R. A conserved 90 nucleotide element mediates translational repression of nanos RNA. Development. 1996 Sep;122(9):2791–2800. doi: 10.1242/dev.122.9.2791. [DOI] [PubMed] [Google Scholar]
  21. Gebauer F., Corona D. F., Preiss T., Becker P. B., Hentze M. W. Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 1999 Nov 1;18(21):6146–6154. doi: 10.1093/emboj/18.21.6146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gray N. K., Wickens M. Control of translation initiation in animals. Annu Rev Cell Dev Biol. 1998;14:399–458. doi: 10.1146/annurev.cellbio.14.1.399. [DOI] [PubMed] [Google Scholar]
  23. Groisman I., Huang Y. S., Mendez R., Cao Q., Theurkauf W., Richter J. D. CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division. Cell. 2000 Oct 27;103(3):435–447. doi: 10.1016/s0092-8674(00)00135-5. [DOI] [PubMed] [Google Scholar]
  24. Hake L. E., Richter J. D. CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell. 1994 Nov 18;79(4):617–627. doi: 10.1016/0092-8674(94)90547-9. [DOI] [PubMed] [Google Scholar]
  25. Havin L., Git A., Elisha Z., Oberman F., Yaniv K., Schwartz S. P., Standart N., Yisraeli J. K. RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization. Genes Dev. 1998 Jun 1;12(11):1593–1598. doi: 10.1101/gad.12.11.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hyatt B. A., Yost H. J. The left-right coordinator: the role of Vg1 in organizing left-right axis formation. Cell. 1998 Apr 3;93(1):37–46. doi: 10.1016/s0092-8674(00)81144-7. [DOI] [PubMed] [Google Scholar]
  27. Iizuka N., Najita L., Franzusoff A., Sarnow P. Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. Mol Cell Biol. 1994 Nov;14(11):7322–7330. doi: 10.1128/mcb.14.11.7322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim-Ha J., Kerr K., Macdonald P. M. Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell. 1995 May 5;81(3):403–412. doi: 10.1016/0092-8674(95)90393-3. [DOI] [PubMed] [Google Scholar]
  29. Kim-Ha J., Smith J. L., Macdonald P. M. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell. 1991 Jul 12;66(1):23–35. doi: 10.1016/0092-8674(91)90136-m. [DOI] [PubMed] [Google Scholar]
  30. King M. L., Zhou Y., Bubunenko M. Polarizing genetic information in the egg: RNA localization in the frog oocyte. Bioessays. 1999 Jul;21(7):546–557. doi: 10.1002/(SICI)1521-1878(199907)21:7<546::AID-BIES3>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  31. Kobayashi H., Minshull J., Ford C., Golsteyn R., Poon R., Hunt T. On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis. J Cell Biol. 1991 Aug;114(4):755–765. doi: 10.1083/jcb.114.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kruys V., Marinx O., Shaw G., Deschamps J., Huez G. Translational blockade imposed by cytokine-derived UA-rich sequences. Science. 1989 Aug 25;245(4920):852–855. doi: 10.1126/science.2672333. [DOI] [PubMed] [Google Scholar]
  33. Lewis T., Gueydan C., Huez G., Toulmé J. J., Kruys V. Mapping of a minimal AU-rich sequence required for lipopolysaccharide-induced binding of a 55-kDa protein on tumor necrosis factor-alpha mRNA. J Biol Chem. 1998 May 29;273(22):13781–13786. doi: 10.1074/jbc.273.22.13781. [DOI] [PubMed] [Google Scholar]
  34. Lie Y. S., Macdonald P. M. Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts. Development. 1999 Nov;126(22):4989–4996. doi: 10.1242/dev.126.22.4989. [DOI] [PubMed] [Google Scholar]
  35. Macdonald P. M., Smibert C. A. Translational regulation of maternal mRNAs. Curr Opin Genet Dev. 1996 Aug;6(4):403–407. doi: 10.1016/s0959-437x(96)80060-8. [DOI] [PubMed] [Google Scholar]
  36. Macdonald P. M. The Drosophila pumilio gene: an unusually long transcription unit and an unusual protein. Development. 1992 Jan;114(1):221–232. doi: 10.1242/dev.114.1.221. [DOI] [PubMed] [Google Scholar]
  37. Marinx O., Bertrand S., Karsenti E., Huez G., Kruys V. Fertilization of Xenopus eggs imposes a complete translational arrest of mRNAs containing 3'UUAUUUAU elements. FEBS Lett. 1994 May 30;345(2-3):107–112. doi: 10.1016/0014-5793(94)00413-7. [DOI] [PubMed] [Google Scholar]
  38. Matthews G., Colman A. A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs. Nucleic Acids Res. 1991 Dec 11;19(23):6405–6412. doi: 10.1093/nar/19.23.6405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Melton D. A. Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes. Nature. 1987 Jul 2;328(6125):80–82. doi: 10.1038/328080a0. [DOI] [PubMed] [Google Scholar]
  40. Mendez R., Murthy K. G., Ryan K., Manley J. L., Richter J. D. Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell. 2000 Nov;6(5):1253–1259. doi: 10.1016/s1097-2765(00)00121-0. [DOI] [PubMed] [Google Scholar]
  41. Mowry K. L. Complex formation between stage-specific oocyte factors and a Xenopus mRNA localization element. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14608–14613. doi: 10.1073/pnas.93.25.14608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mowry K. L., Melton D. A. Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes. Science. 1992 Feb 21;255(5047):991–994. doi: 10.1126/science.1546297. [DOI] [PubMed] [Google Scholar]
  43. Murata Y., Wharton R. P. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell. 1995 Mar 10;80(5):747–756. doi: 10.1016/0092-8674(95)90353-4. [DOI] [PubMed] [Google Scholar]
  44. Murray M. T., Schiller D. L., Franke W. W. Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):11–15. doi: 10.1073/pnas.89.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pain V. M., Patrick T. D., Cox R., Morley S. J. Analysis of translational activity of extracts derived from oocytes and eggs of Xenopus laevis. Methods Mol Biol. 1998;77:195–209. doi: 10.1385/0-89603-397-X:195. [DOI] [PubMed] [Google Scholar]
  46. Rebagliati M. R., Weeks D. L., Harvey R. P., Melton D. A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell. 1985 Oct;42(3):769–777. doi: 10.1016/0092-8674(85)90273-9. [DOI] [PubMed] [Google Scholar]
  47. Sallés F. J., Strickland S. Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR Methods Appl. 1995 Jun;4(6):317–321. doi: 10.1101/gr.4.6.317. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Sheets M. D., Fox C. A., Hunt T., Vande Woude G., Wickens M. The 3'-untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev. 1994 Apr 15;8(8):926–938. doi: 10.1101/gad.8.8.926. [DOI] [PubMed] [Google Scholar]
  50. Smibert C. A., Lie Y. S., Shillinglaw W., Henzel W. J., Macdonald P. M. Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro. RNA. 1999 Dec;5(12):1535–1547. doi: 10.1017/s1355838299991392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Smibert C. A., Wilson J. E., Kerr K., Macdonald P. M. smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo. Genes Dev. 1996 Oct 15;10(20):2600–2609. doi: 10.1101/gad.10.20.2600. [DOI] [PubMed] [Google Scholar]
  52. Sonoda J., Wharton R. P. Recruitment of Nanos to hunchback mRNA by Pumilio. Genes Dev. 1999 Oct 15;13(20):2704–2712. doi: 10.1101/gad.13.20.2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. St Johnston D. The intracellular localization of messenger RNAs. Cell. 1995 Apr 21;81(2):161–170. doi: 10.1016/0092-8674(95)90324-0. [DOI] [PubMed] [Google Scholar]
  54. Stebbins-Boaz B., Cao Q., de Moor C. H., Mendez R., Richter J. D. Maskin is a CPEB-associated factor that transiently interacts with elF-4E. Mol Cell. 1999 Dec;4(6):1017–1027. doi: 10.1016/s1097-2765(00)80230-0. [DOI] [PubMed] [Google Scholar]
  55. Stebbins-Boaz B., Hake L. E., Richter J. D. CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus. EMBO J. 1996 May 15;15(10):2582–2592. [PMC free article] [PubMed] [Google Scholar]
  56. Tannahill D., Melton D. A. Localized synthesis of the Vg1 protein during early Xenopus development. Development. 1989 Aug;106(4):775–785. doi: 10.1242/dev.106.4.775. [DOI] [PubMed] [Google Scholar]
  57. Tautz D., Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989 Aug;98(2):81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]
  58. Walker J., Minshall N., Hake L., Richter J., Standart N. The clam 3' UTR masking element-binding protein p82 is a member of the CPEB family. RNA. 1999 Jan;5(1):14–26. doi: 10.1017/s1355838299981219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wang C., Lehmann R. Nanos is the localized posterior determinant in Drosophila. Cell. 1991 Aug 23;66(4):637–647. doi: 10.1016/0092-8674(91)90110-k. [DOI] [PubMed] [Google Scholar]
  60. Weeks D. L., Melton D. A. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell. 1987 Dec 4;51(5):861–867. doi: 10.1016/0092-8674(87)90109-7. [DOI] [PubMed] [Google Scholar]
  61. Wharton R. P., Sonoda J., Lee T., Patterson M., Murata Y. The Pumilio RNA-binding domain is also a translational regulator. Mol Cell. 1998 May;1(6):863–872. doi: 10.1016/s1097-2765(00)80085-4. [DOI] [PubMed] [Google Scholar]
  62. Wilhelm J. E., Vale R. D., Hegde R. S. Coordinate control of translation and localization of Vg1 mRNA in Xenopus oocytes. Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13132–13137. doi: 10.1073/pnas.97.24.13132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wormington M. Preparation of synthetic mRNAs and analyses of translational efficiency in microinjected Xenopus oocytes. Methods Cell Biol. 1991;36:167–183. doi: 10.1016/s0091-679x(08)60277-0. [DOI] [PubMed] [Google Scholar]
  64. Xu N., Chen C. Y., Shyu A. B. Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay. Mol Cell Biol. 1997 Aug;17(8):4611–4621. doi: 10.1128/mcb.17.8.4611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yisraeli J. K., Melton D. A. The material mRNA Vg1 is correctly localized following injection into Xenopus oocytes. Nature. 1988 Dec 8;336(6199):592–595. doi: 10.1038/336592a0. [DOI] [PubMed] [Google Scholar]
  66. de Moor C. H., Richter J. D. Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA. EMBO J. 1999 Apr 15;18(8):2294–2303. doi: 10.1093/emboj/18.8.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES