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. 1994 Jul 11;22(13):2643–2650. doi: 10.1093/nar/22.13.2643

Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains.

H Jacquemin-Sablon 1, G Triqueneaux 1, S Deschamps 1, M le Maire 1, J Doniger 1, F Dautry 1
PMCID: PMC308222  PMID: 7518919

Abstract

The unr gene was identified as a transcription unit located immediately upstream of N-ras in the genome of several mammalian species. While this genetic organization could be important for the transcriptional regulation of unr and N-ras, the function of the protein product of unr is unknown. unr is ubiquitously expressed and codes for an 85 kDa protein which is not closely related to previously characterized proteins. Nevertheless, a search for protein motifs has indicated the presence of five 'cold shock domains' within unr, a motif present in procaryotic cold shock proteins and in the vertebrate Y box factors. As these proteins have been reported to interact with nucleic acids, we investigated whether unr could bind to some classes of nucleic acids. We report here that unr has a high affinity for single-stranded DNA or RNA and a low affinity for double-stranded nucleic acids. Its low affinity for double-stranded DNA clearly distinguishes unr from the Y box factors. The binding of unr to RNA does not appear to depend upon extended sequence motifs but requires some level of sequence complexity as unr has only a low affinity for most simple polymers including polyA stretches. unr is also characterized by its low affinity for double-stranded and structured RNAs. We further determined that unr is mostly localized in the cytoplasm, and is in part associated with the endoplasmic reticulum. These studies indicate that unr is a novel single-stranded nucleic acid binding protein which is likely to be associated with cytoplasmic mRNA in vivo.

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Selected References

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  1. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boussadia O., Jacquemin-Sablon H., Dautry F. Exon skipping in the expression of the gene immediately upstream of N-ras (unr/NRU). Biochim Biophys Acta. 1993 Feb 20;1172(1-2):64–72. doi: 10.1016/0167-4781(93)90270-n. [DOI] [PubMed] [Google Scholar]
  3. Cohen I., Reynolds W. F. The Xenopus YB3 protein binds the B box element of the class III promoter. Nucleic Acids Res. 1991 Sep 11;19(17):4753–4759. doi: 10.1093/nar/19.17.4753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Didier D. K., Schiffenbauer J., Woulfe S. L., Zacheis M., Schwartz B. D. Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7322–7326. doi: 10.1073/pnas.85.19.7322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doniger J., DiPaolo J. A. Coordinate N-ras mRNA up-regulation with mutational activation in tumorigenic guinea pig cells. Nucleic Acids Res. 1988 Feb 11;16(3):969–980. doi: 10.1093/nar/16.3.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doniger J., Landsman D., Gonda M. A., Wistow G. The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif. New Biol. 1992 Apr;4(4):389–395. [PubMed] [Google Scholar]
  10. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  11. Gait M. J., Karn J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem Sci. 1993 Jul;18(7):255–259. doi: 10.1016/0968-0004(93)90176-n. [DOI] [PubMed] [Google Scholar]
  12. Goldstein J., Pollitt N. S., Inouye M. Major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):283–287. doi: 10.1073/pnas.87.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grant C. E., Deeley R. G. Cloning and characterization of chicken YB-1: regulation of expression in the liver. Mol Cell Biol. 1993 Jul;13(7):4186–4196. doi: 10.1128/mcb.13.7.4186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hasegawa S. L., Doetsch P. W., Hamilton K. K., Martin A. M., Okenquist S. A., Lenz J., Boss J. M. DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs. Nucleic Acids Res. 1991 Sep 25;19(18):4915–4920. doi: 10.1093/nar/19.18.4915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacquemin-Sablon H., Dautry F. Organization of the unr/N-ras locus: characterization of the promoter region of the human unr gene. Nucleic Acids Res. 1992 Dec 11;20(23):6355–6361. doi: 10.1093/nar/20.23.6355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jeffers M., Paciucci R., Pellicer A. Characterization of unr; a gene closely linked to N-ras. Nucleic Acids Res. 1990 Aug 25;18(16):4891–4899. [PMC free article] [PubMed] [Google Scholar]
  17. Kick D., Barrett P., Cummings A., Sommerville J. Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation. Nucleic Acids Res. 1987 May 26;15(10):4099–4109. doi: 10.1093/nar/15.10.4099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kimura A., Israël A., Le Bail O., Kourilsky P. Detailed analysis of the mouse H-2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression. Cell. 1986 Jan 31;44(2):261–272. doi: 10.1016/0092-8674(86)90760-9. [DOI] [PubMed] [Google Scholar]
  19. Kolluri R., Torrey T. A., Kinniburgh A. J. A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif. Nucleic Acids Res. 1992 Jan 11;20(1):111–116. doi: 10.1093/nar/20.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozak M. Regulation of translation in eukaryotic systems. Annu Rev Cell Biol. 1992;8:197–225. doi: 10.1146/annurev.cb.08.110192.001213. [DOI] [PubMed] [Google Scholar]
  21. Kremers P., Beaune P., Cresteil T., de Graeve J., Columelli S., Leroux J. P., Gielen J. E. Cytochrome P-450 monooxygenase activities in human and rat liver microsomes. Eur J Biochem. 1981 Sep 1;118(3):599–606. doi: 10.1111/j.1432-1033.1981.tb05561.x. [DOI] [PubMed] [Google Scholar]
  22. La Teana A., Brandi A., Falconi M., Spurio R., Pon C. L., Gualerzi C. O. Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10907–10911. doi: 10.1073/pnas.88.23.10907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Landsman D. RNP-1, an RNA-binding motif is conserved in the DNA-binding cold shock domain. Nucleic Acids Res. 1992 Jun 11;20(11):2861–2864. doi: 10.1093/nar/20.11.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  26. Murray M. T., Krohne G., Franke W. W. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos. J Cell Biol. 1991 Jan;112(1):1–11. doi: 10.1083/jcb.112.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Neel H., Weil D., Giansante C., Dautry F. In vivo cooperation between introns during pre-mRNA processing. Genes Dev. 1993 Nov;7(11):2194–2205. doi: 10.1101/gad.7.11.2194. [DOI] [PubMed] [Google Scholar]
  28. Nicolaiew N., Triqueneaux G., Dautry F. Organization of the human N-ras locus: characterization of a gene located immediately upstream of N-ras. Oncogene. 1991 May;6(5):721–730. [PubMed] [Google Scholar]
  29. Ozer J., Faber M., Chalkley R., Sealy L. Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif. J Biol Chem. 1990 Dec 25;265(36):22143–22152. [PubMed] [Google Scholar]
  30. Paciucci R., Pellicer A. Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element. Mol Cell Biol. 1991 Mar;11(3):1334–1343. doi: 10.1128/mcb.11.3.1334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Qoronfleh M. W., Debouck C., Keller J. Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli. J Bacteriol. 1992 Dec;174(24):7902–7909. doi: 10.1128/jb.174.24.7902-7909.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ranjan M., Tafuri S. R., Wolffe A. P. Masking mRNA from translation in somatic cells. Genes Dev. 1993 Sep;7(9):1725–1736. doi: 10.1101/gad.7.9.1725. [DOI] [PubMed] [Google Scholar]
  33. Richter J. D., Smith L. D. Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis. J Biol Chem. 1983 Apr 25;258(8):4864–4869. [PubMed] [Google Scholar]
  34. 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]
  35. Sakura H., Maekawa T., Imamoto F., Yasuda K., Ishii S. Two human genes isolated by a novel method encode DNA-binding proteins containing a common region of homology. Gene. 1988 Dec 20;73(2):499–507. doi: 10.1016/0378-1119(88)90514-8. [DOI] [PubMed] [Google Scholar]
  36. Schindelin H., Marahiel M. A., Heinemann U. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature. 1993 Jul 8;364(6433):164–168. doi: 10.1038/364164a0. [DOI] [PubMed] [Google Scholar]
  37. Schnuchel A., Wiltscheck R., Czisch M., Herrler M., Willimsky G., Graumann P., Marahiel M. A., Holak T. A. Structure in solution of the major cold-shock protein from Bacillus subtilis. Nature. 1993 Jul 8;364(6433):169–171. doi: 10.1038/364169a0. [DOI] [PubMed] [Google Scholar]
  38. Tafuri S. R., Familari M., Wolffe A. P. A mouse Y box protein, MSY1, is associated with paternal mRNA in spermatocytes. J Biol Chem. 1993 Jun 5;268(16):12213–12220. [PubMed] [Google Scholar]
  39. Tafuri S. R., Wolffe A. P. Selective recruitment of masked maternal mRNA from messenger ribonucleoprotein particles containing FRGY2 (mRNP4). J Biol Chem. 1993 Nov 15;268(32):24255–24261. [PubMed] [Google Scholar]
  40. Tafuri S. R., Wolffe A. P. Xenopus Y-box transcription factors: molecular cloning, functional analysis and developmental regulation. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9028–9032. doi: 10.1073/pnas.87.22.9028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thorn J. T., Todd A. V., Warrilow D., Watt F., Molloy P. L., Iland H. J. Characterization of the human N-ras promoter region. Oncogene. 1991 Oct;6(10):1843–1850. [PubMed] [Google Scholar]
  42. Weeks K. M., Crothers D. M. Major groove accessibility of RNA. Science. 1993 Sep 17;261(5128):1574–1577. doi: 10.1126/science.7690496. [DOI] [PubMed] [Google Scholar]
  43. Wilhelm J. E., Vale R. D. RNA on the move: the mRNA localization pathway. J Cell Biol. 1993 Oct;123(2):269–274. doi: 10.1083/jcb.123.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Willimsky G., Bang H., Fischer G., Marahiel M. A. Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures. J Bacteriol. 1992 Oct;174(20):6326–6335. doi: 10.1128/jb.174.20.6326-6335.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wistow G. Cold shock and DNA binding. Nature. 1990 Apr 26;344(6269):823–824. doi: 10.1038/344823c0. [DOI] [PubMed] [Google Scholar]
  46. Wolffe A. P., Tafuri S., Ranjan M., Familari M. The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man. New Biol. 1992 Apr;4(4):290–298. [PubMed] [Google Scholar]
  47. Woodbury C. P., Jr, von Hippel P. H. On the determination of deoxyribonucleic acid-protein interaction parameters using the nitrocellulose filter-binding assay. Biochemistry. 1983 Sep 27;22(20):4730–4737. doi: 10.1021/bi00289a018. [DOI] [PubMed] [Google Scholar]

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