Skip to main content
  • Research Article
  • Published:

Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA

Abstract

Induction of Prostaglandin Endoperoxide H Synthase-1 (PGHS-1) gene has been previously documented in a few studies during events such as development and cellular differentiation. However, molecular mechanisms governing the regulation of PGHS-1 gene expression and contributing to changes in protein levels are poorly understood. Using the MEG-01 cell model of PGHS-1 gene induction, our laboratory has previously demonstrated that the 5’UTR and the first two exons of PGHS-1 mRNA had a significant impact on decreasing the translational efficiency of a reporter gene and suggested that the presence of a secondary structure is required for conservation of this activity. This 5’end of PGHS-1 mRNA sequence has also been shown to associate with nucleolin protein. In the current study, we set to investigate the protein composition of the mRNP (messenger ribonucleoprotein) associating with the 5’end of PGHS-1 mRNA and to identify its protein members. RNA/protein binding assays coupled with LC-MS analysis identified serpin B1 and NF45 (nuclear factor 45) proteins as potential members of PGHS-1 mRNP complex. Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members.

Abbreviations

ARRE-2:

antigen receptor response element-2

ATCC:

American Type Culture Collection

DRBP76:

double-stranded RNA-binding protein-76

dsRNA:

double stranded RNA

FBS:

fetal bovine serum

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

IL-2:

interleukin-2

IP:

immunoprecipitation

IRES:

internal ribosome entry site

LC-MS:

liquid chromatography mass-spectrometry

Luc:

luciferase

MKP-1:

mitogen-activated protein kinase phosphatase 1

MNEI:

monocyte/neutrophil elastase inhibitor

mRNP:

messenger ribonucleoprotein

NCL:

nucleolin

NF45:

nuclear factor 45

NF90:

nuclear factor 90

NFAT:

nuclear factor of activated T-cells

ORF:

open reading frame

PG:

prostaglandins

PGHS:

prostaglandin endoperoxide H synthase

PMA:

phorbol 12-myristate 13-acetate

SB1 or serpin B1:

serine protease inhibitor

TPA:

12-O-tetradecanoylphorbol -13- acetate

Tx:

thromboxanes

UTR:

untranslated region

References

  1. Smith, W.L., DeWitt, D.L. and Garavito, R.M. Cyclooxygenases: Structural, cellular, and molecular biology. Annu. Rev. Biochem. 69 (2000) 145–182. DOI: 10.1146/annurev.biochem.69.1.145.

    Article  CAS  PubMed  Google Scholar 

  2. Smith, W.L. and Langenbach, R. Why there are two cyclooxygenase isozymes. J. Clin. Invest. 107 (2001) 1491–1495. DOI: 10.1172/JCI13271.

    Article  CAS  PubMed  Google Scholar 

  3. Parente, L. and Perretti, M. Advances in the pathophysiology of constitutive and inducible cyclooxygenases: Two enzymes in the spotlight. Biochem. Pharmacol. 65 (2003) 153–159.

    Article  CAS  PubMed  Google Scholar 

  4. Smith, C.J., Morrow, J.D., Roberts, L.J. 2nd and Marnett, L.J. Differentiation of monocytoid THP-1 cells with phorbol ester induces expression of prostaglandin endoperoxide synthase-1 (COX-1). Biochem. Biophys. Res. Commun. 192 (1993) 787–793. DOI: 10.1006/bbrc.1993.1483.

    Article  CAS  PubMed  Google Scholar 

  5. Brannon, T.S., North, A.J., Wells, L.B. and Shaul, P.W. Prostacyclin synthesis in ovine pulmonary artery is developmentally regulated by changes in cyclooxygenase-1 gene expression. J. Clin. Invest. 93 (1994) 2230–2235. DOI: 10.1172/JCI117220.

    Article  CAS  PubMed  Google Scholar 

  6. Mroske, C., Plant, M.H., Franks, D.J. and Laneuville, O. Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01. Exp. Hematol. 28 (2000) 411–421.

    Article  CAS  PubMed  Google Scholar 

  7. Plant, M.H. and Laneuville, O. Characterization of a novel transcript of prostaglandin endoperoxide H synthase 1 with a tissue-specific profile of expression. Biochem. J. 344 Pt 3 (1999) 677–685.

    Article  Google Scholar 

  8. Duquette, M. and Laneuville, O. Translational regulation of prostaglandin endoperoxide H synthase-1 mRNA in megakaryocytic MEG-01 cells. specific protein binding to a conserved 20-nucleotide CIS element in the 3’-untranslated region. J. Biol. Chem. 277 (2002) 44631–44637. DOI: 10.1074/jbc.M207007200.

    Article  CAS  PubMed  Google Scholar 

  9. Jiang, Y.J., Lu, B., Choy, P.C. and Hatch, G.M. Regulation of cytosolic phospholipase A2, cyclooxygenase-1 and -2 expression by PMA, TNFalpha, LPS and M-CSF in human monocytes and macrophages. Mol. Cell. Biochem. 246 (2003) 31–38.

    Article  CAS  PubMed  Google Scholar 

  10. Jiang, Y.J., Xu, T.R., Lu, B., Mymin, D., Kroeger, E.A., Dembinski, T., Yang, X., Hatch, G.M. and Choy, P.C. Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells. Biochim. Biophys. Acta. 1633 (2003) 51–60. DOI: S1388198103000726 [pii].

    CAS  PubMed  Google Scholar 

  11. Rocca, B., Morosetti, R., Habib, A., Maggiano, N., Zassadowski, F., Ciabattoni, G., Chomienne, C., Papp, B. and Ranelletti, F.O. Cyclooxygenase-1, but not -2, is upregulated in NB4 leukemic cells and human primary promyelocytic blasts during differentiation. Leukemia 18 (2004) 1373–1379. DOI: 10.1038/sj.leu.2403407 [doi]; 2403407 [pii].

    Article  CAS  PubMed  Google Scholar 

  12. Schneider, N., Lanz, S., Ramer, R., Schaefer, D. and Goppelt-Struebe, M. Up-regulation of cyclooxygenase-1 in neuroblastoma cell lines by retinoic acid and corticosteroids. J. Neurochem. 77 (2001) 416–424.

    Article  CAS  PubMed  Google Scholar 

  13. Mullol, J., Fernandez-Morata, J.C., Roca-Ferrer, J., Pujols, L., Xaubet, A., Benitez, P. and Picado, C. Cyclooxygenase 1 and cyclooxygenase 2 expression is abnormally regulated in human nasal polyps. J. Allergy Clin. Immunol. 109 (2002) 824–830. DOI: S0091674902962603 [pii].

    Article  CAS  PubMed  Google Scholar 

  14. Bunimov, N., Smith, J.E., Gosselin, D. and Laneuville, O. Translational regulation of PGHS-1 mRNA: 5’ untranslated region and first two exons conferring negative regulation. Biochim. Biophys. Acta. 1769 (2007) 92–105. DOI: 10.1016/j.bbaexp.2007.01.004.

    CAS  PubMed  Google Scholar 

  15. Ghisolfi-Nieto, L., Joseph, G., Puvion-Dutilleul, F., Amalric, F. and Bouvet, P. Nucleolin is a sequence-specific RNA-binding protein: Characterization of targets on pre-ribosomal RNA. J. Mol. Biol. 260 (1996) 34–53. DOI: S0022-2836(96)90380-0 [pii]; 10.1006/jmbi. 1996.0380 [doi].

    Article  CAS  PubMed  Google Scholar 

  16. Moore, M.J. From birth to death: The complex lives of eukaryotic mRNAs. Science 309 (2005) 1514–1518. DOI: 10.1126/science. 1111443.

    Article  CAS  PubMed  Google Scholar 

  17. Holcik, M. and Pestova, T.V. Translation mechanism and regulation: Old players, new concepts. meeting on translational control and non-coding RNA. EMBO Rep. 8 (2007) 639–643. DOI: 10.1038/sj.embor. 7400988.

    Article  CAS  PubMed  Google Scholar 

  18. Mili, S. and Steitz, J.A. Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses. RNA. 10 (2004) 1692–1694. DOI: 10.1261/rna.7151404.

    Article  CAS  PubMed  Google Scholar 

  19. Niranjanakumari, S., Lasda, E., Brazas, R. and Garcia-Blanco, M.A. Reversible cross-linking combined with immunoprecipitation to study RNAprotein interactions in vivo. Methods 26 (2002) 182–190. DOI: 10.1016/S1046-2023(02)00021-X.

    Article  CAS  PubMed  Google Scholar 

  20. Ule, J., Jensen, K., Mele, A. and Darnell, R.B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods 37 (2005) 376–386. DOI: 10.1016/j.ymeth.2005.07.018.

    Article  CAS  PubMed  Google Scholar 

  21. Srisawat, C. and Engelke, D.R. Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins. RNA 7 (2001) 632–641.

    Article  CAS  PubMed  Google Scholar 

  22. Srisawat, C. and Engelke, D.R. RNA affinity tags for purification of RNAs and ribonucleoprotein complexes. Methods 26 (2002) 156–161. DOI: 10.1016/S1046-2023(02)00018-X.

    Article  CAS  PubMed  Google Scholar 

  23. Walker, S.C., Scott, F.H., Srisawat, C. and Engelke, D.R. RNA affinity tags for the rapid purification and investigation of RNAs and RNA-protein complexes. Methods Mol. Biol. 488 (2008) 23–40. DOI: 10.1007/978-1-60327-475-3_3.

    Article  CAS  PubMed  Google Scholar 

  24. Johnson, R.F., McCarthy, S.E., Godlewski, P.J. and Harty, R.N. Ebola virus VP35-VP40 interaction is sufficient for packaging 3E-5E minigenome RNA into virus-like particles. J. Virol. 80 (2006) 5135–5144. DOI: 10.1128/JVI.01857-05.

    Article  CAS  PubMed  Google Scholar 

  25. Trinkle-Mulcahy, L., Boulon, S., Lam, Y.W., Urcia, R., Boisvert, F.M., Vandermoere, F., Morrice, N.A., Swift, S., Rothbauer, U., Leonhardt, H. and Lamond, A. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell Biol. 183 (2008) 223–239. DOI: 10.1083/jcb.200805092.

    Article  CAS  PubMed  Google Scholar 

  26. Satoh, M., Shaheen, V.M., Kao, P.N., Okano, T., Shaw, M., Yoshida, H., Richards, H.B. and Reeves, W.H. Autoantibodies define a family of proteins with conserved double-stranded RNA-binding domains as well as DNA binding activity. J. Biol. Chem. 274 (1999) 34598–34604.

    Article  CAS  PubMed  Google Scholar 

  27. Saunders, L.R., Perkins, D.J., Balachandran, S., Michaels, R., Ford, R., Mayeda, A. and Barber, G.N. Characterization of two evolutionarily conserved, alternatively spliced nuclear phosphoproteins, NFAR-1 and -2, that function in mRNA processing and interact with the double-stranded RNA-dependent protein kinase, PKR. J. Biol. Chem. 276 (2001) 32300–32312. DOI: 10.1074/jbc.M104207200.

    Article  CAS  PubMed  Google Scholar 

  28. Reichman, T.W., Muniz, L.C. and Mathews, M.B. The RNA binding protein nuclear factor 90 functions as both a positive and negative regulator of gene expression in mammalian cells. Mol. Cell. Biol. 22 (2002) 343–356.

    Article  CAS  PubMed  Google Scholar 

  29. Remold-O’Donnell, E., Nixon, J.C. and Rose, R.M. Elastase inhibitor. characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils. J. Exp. Med. 169 (1989) 1071–1086.

    Article  PubMed  Google Scholar 

  30. Cooley, J., Takayama, T.K., Shapiro, S.D., Schechter, N.M. and Remold-O’Donnell, E. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry. 40 (2001) 15762–15770.

    Article  CAS  PubMed  Google Scholar 

  31. Gettins, P. G. W., Patson, P. and Olson, S. Serpins: structure, function and biology, 1st Edition edition, Landes Bioscience, 1996.

  32. Zeng, W., Silverman, G.A. and Remold-O’Donnell, E. Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an ovserpin family gene. Gene 213 (1998) 179–187.

    Article  CAS  PubMed  Google Scholar 

  33. Bird, C.H., Blink, E.J., Hirst, C.E., Buzza, M.S., Steele, P.M., Sun, J., Jans, D.A. and Bird, P.I. Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1. Mol. Cell. Biol. 21 (2001) 5396–5407. DOI: 10.1128/MCB.21.16.5396-5407.2001.

    Article  CAS  PubMed  Google Scholar 

  34. Popova, E.Y., Claxton, D.F., Lukasova, E., Bird, P.I. and Grigoryev, S.A. Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood. Exp. Hematol. 34 (2006) 453–462. DOI: 10.1016/j.exphem.2006.01.003.

    Article  CAS  PubMed  Google Scholar 

  35. Missen, M.A., Haylock, D., Whitty, G., Medcalf, R.L. and Coughlin, P.B. Stage specific gene expression of serpins and their cognate proteases during myeloid differentiation. Br. J. Haematol. 135 (2006) 715–724. DOI: 10.1111/j.1365-2141.2006.06360.x.

    Article  CAS  PubMed  Google Scholar 

  36. Kao, P.N., Chen, L., Brock, G., Ng, J., Kenny, J., Smith, A.J. and Corthesy, B. Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90. J. Biol. Chem. 269 (1994) 20691–20699.

    CAS  PubMed  Google Scholar 

  37. Corthesy, B. and Kao, P.N. Purification by DNA affinity chromatography of two polypeptides that contact the NF-AT DNA binding site in the interleukin 2 promoter. J. Biol. Chem. 269 (1994) 20682–20690.

    CAS  PubMed  Google Scholar 

  38. Marcoulatos, P., Koussidis, G., Mamuris, Z., Velissariou, V. and Vamvakopoulos, N.C. Mapping interleukin enhancer binding factor 2 gene (ILF2) to human chromosome 1 (1q11-qter and 1p11-p12) by polymerase chain reaction amplification of human-rodent somatic cell hybrid DNA templates. J. Interferon Cytokine Res. 16 (1996) 1035–1038.

    CAS  PubMed  Google Scholar 

  39. Guan, D., Altan-Bonnet, N., Parrott, A.M., Arrigo, C.J., Li, Q., Khaleduzzaman, M., Li, H., Lee, C.G., Pe’ery, T. and Mathews, M.B. Nuclear factor 45 (NF45) is a regulatory subunit of complexes with NF90/110 involved in mitotic control. Mol. Cell. Biol. 28 (2008) 4629–4641. DOI: 10.1128/MCB.00120-08.

    Article  CAS  PubMed  Google Scholar 

  40. Merrill, M.K. and Gromeier, M. The double-stranded RNA binding protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type 2 internal ribosome entry site. J. Virol. 80 (2006) 6936–6942. DOI: 10.1128/JVI.00243-06.

    Article  CAS  PubMed  Google Scholar 

  41. Shim, J., Lim, H., Yates, J. and Karin, M. Nuclear export of NF90 is required for interleukin-2 mRNA stabilization. Mol. Cell. 10 (2002) 1331–1344.

    Article  CAS  PubMed  Google Scholar 

  42. Kuwano, Y., Kim, H.H., Abdelmohsen, K., Pullmann, R.,Jr, Martindale, J.L., Yang, X. and Gorospe, M. MKP-1 mRNA stabilization and translational control by RNA-binding proteins HuR and NF90. Mol. Cell. Biol. 28 (2008) 4562–4575. DOI: 10.1128/MCB.00165-08.

    Article  CAS  PubMed  Google Scholar 

  43. Xu, Y.H. and Grabowski, G.A. Molecular cloning and characterization of a translational inhibitory protein that binds to coding sequences of human acid beta-glucosidase and other mRNAs. Mol. Genet. Metab. 68 (1999) 441–454. DOI: 10.1006/mgme.1999.2934.

    Article  CAS  PubMed  Google Scholar 

  44. Maruo, N., Kobayashi, Y., Horiuchi, H., Kondo, M. and Fujita, S. Histochemical study on the maturation of human megakaryocytes using microfluorometry. Histochemistry 97 (1992) 141–145.

    Article  CAS  PubMed  Google Scholar 

  45. Yanagida, M., Shimamoto, A., Nishikawa, K., Furuichi, Y., Isobe, T. and Takahashi, N. Isolation and proteomic characterization of the major proteins of the nucleolin-binding ribonucleoprotein complexes. Proteomics 1 (2001) 1390–1404. DOI: 2-Z.

    Article  CAS  PubMed  Google Scholar 

  46. Chen, C.Y., Gherzi, R., Andersen, J.S., Gaietta, G., Jurchott, K., Royer, H.D., Mann, M. and Karin, M. Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev. 14 (2000) 1236–1248.

    CAS  PubMed  Google Scholar 

  47. Raslova, H., Kauffmann, A., Sekkaï, D., Ripoche, H., Larbret, F., Robert, T., Le Roux, D.T., Kroemer, G., Debili, N., Dessen, P., Lazar, V. and Vainchenker, W. Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach. Blood 109 (2007) 3225–3234. DOI 10.1182/blood-2006-07-037838.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Odette Laneuville.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bunimov, N., Laneuville, O. Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA. Cell Mol Biol Lett 15, 196–214 (2010). https://doi.org/10.2478/s11658-010-0005-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-010-0005-5

Key words