Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Aug 1;25(15):3088–3094. doi: 10.1093/nar/25.15.3088

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes.

G Pont-Kingdon 1, E Chi 1, S Christensen 1, D Carroll 1
PMCID: PMC146839  PMID: 9224609

Abstract

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.

Full Text

The Full Text of this article is available as a PDF (148.4 KB).

Selected References

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

  1. Alber T. Structure of the leucine zipper. Curr Opin Genet Dev. 1992 Apr;2(2):205–210. doi: 10.1016/s0959-437x(05)80275-8. [DOI] [PubMed] [Google Scholar]
  2. Bass B. L., Hurst S. R., Singer J. D. Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs. Curr Biol. 1994 Apr 1;4(4):301–314. doi: 10.1016/s0960-9822(00)00069-5. [DOI] [PubMed] [Google Scholar]
  3. Bouhidel K., Terzian C., Pinon H. The full-length transcript of the I factor, a LINE element of Drosophila melanogaster, is a potential bicistronic RNA messenger. Nucleic Acids Res. 1994 Jun 25;22(12):2370–2374. doi: 10.1093/nar/22.12.2370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bouvet P., Wolffe A. P. A role for transcription and FRGY2 in masking maternal mRNA within Xenopus oocytes. Cell. 1994 Jun 17;77(6):931–941. doi: 10.1016/0092-8674(94)90141-4. [DOI] [PubMed] [Google Scholar]
  5. Braddock M., Muckenthaler M., White M. R., Thorburn A. M., Sommerville J., Kingsman A. J., Kingsman S. M. Intron-less RNA injected into the nucleus of Xenopus oocytes accesses a regulated translation control pathway. Nucleic Acids Res. 1994 Dec 11;22(24):5255–5264. doi: 10.1093/nar/22.24.5255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Branciforte D., Martin S. L. Developmental and cell type specificity of LINE-1 expression in mouse testis: implications for transposition. Mol Cell Biol. 1994 Apr;14(4):2584–2592. doi: 10.1128/mcb.14.4.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bukrinsky M. I., Haggerty S., Dempsey M. P., Sharova N., Adzhubel A., Spitz L., Lewis P., Goldfarb D., Emerman M., Stevenson M. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993 Oct 14;365(6447):666–669. doi: 10.1038/365666a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Darnbrough C. H., Ford P. J. Identification in Xenopus laevis of a class of oocyte-specific proteins bound to messenger RNA. Eur J Biochem. 1981 Jan;113(3):415–424. doi: 10.1111/j.1432-1033.1981.tb05081.x. [DOI] [PubMed] [Google Scholar]
  9. Dearsly A. L., Johnson R. M., Barrett P., Sommerville J. Identification of a 60-kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes. Eur J Biochem. 1985 Jul 1;150(1):95–103. doi: 10.1111/j.1432-1033.1985.tb08993.x. [DOI] [PubMed] [Google Scholar]
  10. Deragon J. M., Sinnett D., Labuda D. Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1. EMBO J. 1990 Oct;9(10):3363–3368. doi: 10.1002/j.1460-2075.1990.tb07537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deschamps S., Viel A., Garrigos M., Denis H., le Maire M. mRNP4, a major mRNA-binding protein from Xenopus oocytes is identical to transcription factor FRG Y2. J Biol Chem. 1992 Jul 15;267(20):13799–13802. [PubMed] [Google Scholar]
  12. Eickbush T. H. Transposing without ends: the non-LTR retrotransposable elements. New Biol. 1992 May;4(5):430–440. [PubMed] [Google Scholar]
  13. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Feng Q., Moran J. V., Kazazian H. H., Jr, Boeke J. D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell. 1996 Nov 29;87(5):905–916. doi: 10.1016/s0092-8674(00)81997-2. [DOI] [PubMed] [Google Scholar]
  16. Garrett J. E., Carroll D. Tx1: a transposable element from Xenopus laevis with some unusual properties. Mol Cell Biol. 1986 Mar;6(3):933–941. doi: 10.1128/mcb.6.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garrett J. E., Knutzon D. S., Carroll D. Composite transposable elements in the Xenopus laevis genome. Mol Cell Biol. 1989 Jul;9(7):3018–3027. doi: 10.1128/mcb.9.7.3018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hohjoh H., Singer M. F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. EMBO J. 1996 Feb 1;15(3):630–639. [PMC free article] [PubMed] [Google Scholar]
  19. Holmes S. E., Singer M. F., Swergold G. D. Studies on p40, the leucine zipper motif-containing protein encoded by the first open reading frame of an active human LINE-1 transposable element. J Biol Chem. 1992 Oct 5;267(28):19765–19768. [PubMed] [Google Scholar]
  20. Ilves H., Kahre O., Speek M. Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting. Mol Cell Biol. 1992 Sep;12(9):4242–4248. doi: 10.1128/mcb.12.9.4242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kolosha V. O., Martin S. L. Polymorphic sequences encoding the first open reading frame protein from LINE-1 ribonucleoprotein particles. J Biol Chem. 1995 Feb 10;270(6):2868–2873. doi: 10.1074/jbc.270.6.2868. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Krieg P., Strachan R., Wallis E., Tabe L., Colman A. Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes. J Mol Biol. 1984 Dec 15;180(3):615–643. doi: 10.1016/0022-2836(84)90030-5. [DOI] [PubMed] [Google Scholar]
  24. Lu Z., Templer M., Nielsen B. L. Rapid method for recovery of DNA from agarose gels. Biotechniques. 1994 Mar;16(3):400–402. [PubMed] [Google Scholar]
  25. Luan D. D., Korman M. H., Jakubczak J. L., Eickbush T. H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell. 1993 Feb 26;72(4):595–605. doi: 10.1016/0092-8674(93)90078-5. [DOI] [PubMed] [Google Scholar]
  26. Martin S. L., Branciforte D. Synchronous expression of LINE-1 RNA and protein in mouse embryonal carcinoma cells. Mol Cell Biol. 1993 Sep;13(9):5383–5392. doi: 10.1128/mcb.13.9.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martin S. L. LINEs. Curr Opin Genet Dev. 1991 Dec;1(4):505–508. doi: 10.1016/s0959-437x(05)80199-6. [DOI] [PubMed] [Google Scholar]
  28. Martin S. L. Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol. 1991 Sep;11(9):4804–4807. doi: 10.1128/mcb.11.9.4804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maryon E., Carroll D. Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes. Mol Cell Biol. 1989 Nov;9(11):4862–4871. doi: 10.1128/mcb.9.11.4862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mathias S. L., Scott A. F., Kazazian H. H., Jr, Boeke J. D., Gabriel A. Reverse transcriptase encoded by a human transposable element. Science. 1991 Dec 20;254(5039):1808–1810. doi: 10.1126/science.1722352. [DOI] [PubMed] [Google Scholar]
  31. McMillan J. P., Singer M. F. Translation of the human LINE-1 element, L1Hs. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11533–11537. doi: 10.1073/pnas.90.24.11533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moran J. V., Holmes S. E., Naas T. P., DeBerardinis R. J., Boeke J. D., Kazazian H. H., Jr High frequency retrotransposition in cultured mammalian cells. Cell. 1996 Nov 29;87(5):917–927. doi: 10.1016/s0092-8674(00)81998-4. [DOI] [PubMed] [Google Scholar]
  34. Ogretmen B., Ratajczak H., Kats A., Stark B. C., Gendel S. M. Effects of staining of RNA with ethidium bromide before electrophoresis on performance of northern blots. Biotechniques. 1993 Jun;14(6):932–935. [PubMed] [Google Scholar]
  35. Roe T., Reynolds T. C., Yu G., Brown P. O. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 1993 May;12(5):2099–2108. doi: 10.1002/j.1460-2075.1993.tb05858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sommerville J., Ladomery M. Masking of mRNA by Y-box proteins. FASEB J. 1996 Mar;10(4):435–443. doi: 10.1096/fasebj.10.4.8647342. [DOI] [PubMed] [Google Scholar]
  38. Tsang S. S., Yin X., Guzzo-Arkuran C., Jones V. S., Davison A. J. Loss of resolution in gel electrophoresis of RNA: a problem associated with the presence of formaldehyde gradients. Biotechniques. 1993 Mar;14(3):380–381. [PubMed] [Google Scholar]
  39. Urnes M. S., Carroll D. Amylase synthesis as a simple model system for translation and hybrid arrest in Xenopus oocytes. Gene. 1990 Nov 15;95(2):267–274. doi: 10.1016/0378-1119(90)90370-7. [DOI] [PubMed] [Google Scholar]
  40. Wallace R. A., Jared D. W., Dumont J. N., Sega M. W. Protein incorporation by isolated amphibian oocytes. 3. Optimum incubation conditions. J Exp Zool. 1973 Jun;184(3):321–333. doi: 10.1002/jez.1401840305. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Zimmerly S., Guo H., Perlman P. S., Lambowitz A. M. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell. 1995 Aug 25;82(4):545–554. doi: 10.1016/0092-8674(95)90027-6. [DOI] [PubMed] [Google Scholar]
  43. von Schwedler U., Kornbluth R. S., Trono D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6992–6996. doi: 10.1073/pnas.91.15.6992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES