Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 May 1;339(Pt 3):695–703.

Analysis of RNA-protein interactions of mouse liver cytochrome P4502A5 mRNA.

A Tilloy-Ellul 1, F Raffalli-Mathieu 1, M A Lang 1
PMCID: PMC1220206  PMID: 10215609

Abstract

In our previous studies we have identified a 37/39 kDa, pyrazole-inducible, cytochrome P4502A5 (CYP2A5) mRNA binding protein and provided evidence that it may play a role in the stabilization and processing of the RNA [Geneste, Rafalli and Lang (1996) Biochem. J. 313, 1029-1037; Thulke-Gross, Hergenhahn, Tilloy-Ellul, Lang and Bartsch (1998) Biochem. J. 331, 473-481]. Details of the RNA-protein interactions are, however, not known. In this report we have performed an analysis of the interaction between the CYP2A5 mRNA and the 37/39 kDa protein. With UV-cross linking experiments, using RNA probes corresponding to various parts of the CYP2A5 mRNA, and with antisense oligonucleotides complementary to certain areas of the 3'-untranslated region (3'UTR), we could map the primary binding site to the tip of a 71 nt hair-pin loop at the 3'-UTR. This analysis also showed that the protein may have more than one site of interaction with the RNA and/or that, within the binding region, there could be more than one protein molecule binding to the RNA. Analysis of the probable conformations of the various probes used in the UV cross-linking experiments, in combination with the estimated binding affinities of the protein to the different probes, suggests that important factors in the high-affinity binding are the UAG triplet flanked by GA-rich sequences at the tip of the hair-pin loop, in addition to the conformation of the loop itself. Within the binding region, similarities with known binding sites of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in other RNA molecules were revealed by sequence alignment analysis. Moreover, competition experiments with an oligoribonucleotide corresponding to a known high-affinity binding site of hnRNP A1, and immunoprecipitation of the UV cross-linked 37/39 kDa complex showed that the protein binding to the CYP2A5 mRNA could be hnRNP A1 or its close analogue. It was also shown that the 37/39 kDa protein binds with less affinity to CYP2A4 mRNA than to CYP2A5 mRNA. This is in accordance with experiments characterizing the binding site, since these two otherwise highly homologous genes are kown to have a three nucleotide difference within the region important for the high binding affinity. Since the response of CYP2A4 to pyrazole is known to be weak, as compared with CYP2A5, this observation provides further evidence for a regulatory role of the 37/39 kDa protein in CYP2A5 mRNA metabolism.

Full Text

The Full Text of this article is available as a PDF (210.5 KB).

Selected References

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

  1. Abdul-Manan N., O'Malley S. M., Williams K. R. Origins of binding specificity of the A1 heterogeneous nuclear ribonucleoprotein. Biochemistry. 1996 Mar 19;35(11):3545–3554. doi: 10.1021/bi952298p. [DOI] [PubMed] [Google Scholar]
  2. Abdul-Manan N., Williams K. R. hnRNP A1 binds promiscuously to oligoribonucleotides: utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding. Nucleic Acids Res. 1996 Oct 15;24(20):4063–4070. doi: 10.1093/nar/24.20.4063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Agellon L. B., Cheema S. K. The 3'-untranslated region of the mouse cholesterol 7alpha-hydroxylase mRNA contains elements responsive to post-transcriptional regulation by bile acids. Biochem J. 1997 Dec 1;328(Pt 2):393–399. doi: 10.1042/bj3280393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aida K., Negishi M. Posttranscriptional regulation of coumarin 7-hydroxylase induction by xenobiotics in mouse liver: mRNA stabilization by pyrazole. Biochemistry. 1991 Aug 13;30(32):8041–8045. doi: 10.1021/bi00246a023. [DOI] [PubMed] [Google Scholar]
  5. Burd C. G., Dreyfuss G. RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. EMBO J. 1994 Mar 1;13(5):1197–1204. doi: 10.1002/j.1460-2075.1994.tb06369.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cartegni L., Maconi M., Morandi E., Cobianchi F., Riva S., Biamonti G. hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins. J Mol Biol. 1996 Jun 14;259(3):337–348. doi: 10.1006/jmbi.1996.0324. [DOI] [PubMed] [Google Scholar]
  7. Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
  8. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  9. Geneste O., Raffalli F., Lang M. A. Identification and characterization of a 44 kDa protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization. Biochem J. 1996 Feb 1;313(Pt 3):1029–1037. doi: 10.1042/bj3131029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hahnemann B., Salonpä P., Pasanen M., Mäenpä J., Honkakoski P., Juvonen R., Lang M. A., Pelkonen O., Raunio H. Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression. Biochem J. 1992 Aug 15;286(Pt 1):289–294. doi: 10.1042/bj2860289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hamilton B. J., Nagy E., Malter J. S., Arrick B. A., Rigby W. F. Association of heterogeneous nuclear ribonucleoprotein A1 and C proteins with reiterated AUUUA sequences. J Biol Chem. 1993 Apr 25;268(12):8881–8887. [PubMed] [Google Scholar]
  12. Honkakoski P., Kojo A., Lang M. A. Regulation of the mouse liver cytochrome P450 2B subfamily by sex hormones and phenobarbital. Biochem J. 1992 Aug 1;285(Pt 3):979–983. doi: 10.1042/bj2850979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  14. Li H. P., Zhang X., Duncan R., Comai L., Lai M. M. Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9544–9549. doi: 10.1073/pnas.94.18.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  17. Munroe S. H., Dong X. F. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895–899. doi: 10.1073/pnas.89.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nadler S. G., Merrill B. M., Roberts W. J., Keating K. M., Lisbin M. J., Barnett S. F., Wilson S. H., Williams K. R. Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry. 1991 Mar 19;30(11):2968–2976. doi: 10.1021/bi00225a034. [DOI] [PubMed] [Google Scholar]
  19. Nelson D. R., Kamataki T., Waxman D. J., Guengerich F. P., Estabrook R. W., Feyereisen R., Gonzalez F. J., Coon M. J., Gunsalus I. C., Gotoh O. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 1993 Jan-Feb;12(1):1–51. doi: 10.1089/dna.1993.12.1. [DOI] [PubMed] [Google Scholar]
  20. Pellinen P., Stenbäck F., Rautio A., Pelkonen O., Lang M., Pasanen M. Response of mouse liver coumarin 7-hydroxylase activity to hepatotoxins: dependence on strain and agent and comparison to other monooxygenases. Naunyn Schmiedebergs Arch Pharmacol. 1993 Oct;348(4):435–443. doi: 10.1007/BF00171345. [DOI] [PubMed] [Google Scholar]
  21. Porter T. D., Coon M. J. Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem. 1991 Jul 25;266(21):13469–13472. [PubMed] [Google Scholar]
  22. Raffalli-Mathieu F., Geneste O., Lang M. A. Characterization of two nuclear proteins that interact with cytochrome P-450 1A2 mRNA. Regulation of RNA binding and possible role in the expression of the Cyp1a2 gene. Eur J Biochem. 1997 Apr 1;245(1):17–24. doi: 10.1111/j.1432-1033.1997.00017.x. [DOI] [PubMed] [Google Scholar]
  23. Svitkin Y. V., Ovchinnikov L. P., Dreyfuss G., Sonenberg N. General RNA binding proteins render translation cap dependent. EMBO J. 1996 Dec 16;15(24):7147–7155. [PMC free article] [PubMed] [Google Scholar]
  24. Thulke-Gross M., Hergenhahn M., Tilloy-Ellul A., Lang M., Bartsch H. Pyrazole-inducible proteins in DBA/2 mouse liver bind with high affinity to the 3'-untranslated regions of the mRNAs of coumarin hydroxylase (CYP2A5) and c-jun. Biochem J. 1998 Apr 15;331(Pt 2):473–481. doi: 10.1042/bj3310473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Visa N., Alzhanova-Ericsson A. T., Sun X., Kiseleva E., Björkroth B., Wurtz T., Daneholt B. A pre-mRNA-binding protein accompanies the RNA from the gene through the nuclear pores and into polysomes. Cell. 1996 Jan 26;84(2):253–264. doi: 10.1016/s0092-8674(00)80980-0. [DOI] [PubMed] [Google Scholar]
  26. Wu D., Cederbaum A. I. Expression of cytochrome P4502E1 in rat fetal hepatocyte culture. Mol Pharmacol. 1996 May;49(5):802–807. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES