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. 2010 Jan 28;15(2):196–214. doi: 10.2478/s11658-010-0005-5

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

Natalia Bunimov 1, Odette Laneuville 1,✉
PMCID: PMC6275937  PMID: 20112001

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.

Electronic Supplementary Material

Supplementary material is available for this article at 10.2478/s11658-010-0005-5 and is accessible for authorized users.

Key words: Prostaglandin endoperoxide H synthase-1, Cyclooxygenase-1, MEG-01, Megakaryoblastic cells, Untranslated region, Open reading frame, Messenger ribonucleoprotein, Serpin B1, Nuclear factor 45, Nuclear factor 90

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Abbreviations used

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., Garavito R.M. Cyclooxygenases: Structural, cellular, and molecular biology. Annu. Rev. Biochem. 2000;69:145–182. doi: 10.1146/annurev.biochem.69.1.145. [DOI] [PubMed] [Google Scholar]
  • 2.Smith W.L., Langenbach R. Why there are two cyclooxygenase isozymes. J. Clin. Invest. 2001;107:1491–1495. doi: 10.1172/JCI13271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Parente L., Perretti M. Advances in the pathophysiology of constitutive and inducible cyclooxygenases: Two enzymes in the spotlight. Biochem. Pharmacol. 2003;65:153–159. doi: 10.1016/S0006-2952(02)01422-3. [DOI] [PubMed] [Google Scholar]
  • 4.Smith C.J., Morrow J.D., Roberts L.J., 2nd, 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. 1993;192:787–793. doi: 10.1006/bbrc.1993.1483. [DOI] [PubMed] [Google Scholar]
  • 5.Brannon T.S., North A.J., Wells L.B., Shaul P.W. Prostacyclin synthesis in ovine pulmonary artery is developmentally regulated by changes in cyclooxygenase-1 gene expression. J. Clin. Invest. 1994;93:2230–2235. doi: 10.1172/JCI117220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Mroske C., Plant M.H., Franks D.J., Laneuville O. Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01. Exp. Hematol. 2000;28:411–421. doi: 10.1016/S0301-472X(00)00125-9. [DOI] [PubMed] [Google Scholar]
  • 7.Plant M.H., Laneuville O. Characterization of a novel transcript of prostaglandin endoperoxide H synthase 1 with a tissue-specific profile of expression. Biochem. J. 1999;344Pt3:677–685. doi: 10.1042/0264-6021:3440677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Duquette M., 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. 2002;277:44631–44637. doi: 10.1074/jbc.M207007200. [DOI] [PubMed] [Google Scholar]
  • 9.Jiang Y.J., Lu B., Choy P.C., 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. 2003;246:31–38. doi: 10.1023/A:1023495626568. [DOI] [PubMed] [Google Scholar]
  • 10.Jiang Y.J., Xu T.R., Lu B., Mymin D., Kroeger E.A., Dembinski T., Yang X., Hatch G.M., Choy P.C. Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells. Biochim. Biophys. Acta. 2003;1633:51–60. doi: 10.1016/s1388-1981(03)00072-6. [DOI] [PubMed] [Google Scholar]
  • 11.Rocca B., Morosetti R., Habib A., Maggiano N., Zassadowski F., Ciabattoni G., Chomienne C., Papp B., Ranelletti F.O. Cyclooxygenase-1, but not -2, is upregulated in NB4 leukemic cells and human primary promyelocytic blasts during differentiation. Leukemia. 2004;18:1373–1379. doi: 10.1038/sj.leu.2403407. [DOI] [PubMed] [Google Scholar]
  • 12.Schneider N., Lanz S., Ramer R., Schaefer D., Goppelt-Struebe M. Up-regulation of cyclooxygenase-1 in neuroblastoma cell lines by retinoic acid and corticosteroids. J. Neurochem. 2001;77:416–424. doi: 10.1046/j.1471-4159.2001.00264.x. [DOI] [PubMed] [Google Scholar]
  • 13.Mullol J., Fernandez-Morata J.C., Roca-Ferrer J., Pujols L., Xaubet A., Benitez P., Picado C. Cyclooxygenase 1 and cyclooxygenase 2 expression is abnormally regulated in human nasal polyps. J. Allergy Clin. Immunol. 2002;109:824–830. doi: 10.1067/mai.2002.123534. [DOI] [PubMed] [Google Scholar]
  • 14.Bunimov N., Smith J.E., Gosselin D., Laneuville O. Translational regulation of PGHS-1 mRNA: 5’ untranslated region and first two exons conferring negative regulation. Biochim. Biophys. Acta. 2007;1769:92–105. doi: 10.1016/j.bbaexp.2007.01.004. [DOI] [PubMed] [Google Scholar]
  • 15.Ghisolfi-Nieto L., Joseph G., Puvion-Dutilleul F., Amalric F., Bouvet P. Nucleolin is a sequence-specific RNA-binding protein: Characterization of targets on pre-ribosomal RNA. J. Mol. Biol. 1996;260:34–53. doi: 10.1006/jmbi.1996.0380. [DOI] [PubMed] [Google Scholar]
  • 16.Moore M.J. From birth to death: The complex lives of eukaryotic mRNAs. Science. 2005;309:1514–1518. doi: 10.1126/science.1111443. [DOI] [PubMed] [Google Scholar]
  • 17.Holcik M., Pestova T.V. Translation mechanism and regulation: Old players, new concepts. meeting on translational control and non-coding RNA. EMBO Rep. 2007;8:639–643. doi: 10.1038/sj.embor.7400988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mili S., Steitz J.A. Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses. RNA. 2004;10:1692–1694. doi: 10.1261/rna.7151404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Niranjanakumari S., Lasda E., Brazas R., Garcia-Blanco M.A. Reversible cross-linking combined with immunoprecipitation to study RNAprotein interactions in vivo. Methods. 2002;26:182–190. doi: 10.1016/S1046-2023(02)00021-X. [DOI] [PubMed] [Google Scholar]
  • 20.Ule J., Jensen K., Mele A., Darnell R.B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 2005;37:376–386. doi: 10.1016/j.ymeth.2005.07.018. [DOI] [PubMed] [Google Scholar]
  • 21.Srisawat C., Engelke D.R. Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins. RNA. 2001;7:632–641. doi: 10.1017/S135583820100245X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Srisawat C., Engelke D.R. RNA affinity tags for purification of RNAs and ribonucleoprotein complexes. Methods. 2002;26:156–161. doi: 10.1016/S1046-2023(02)00018-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Walker S.C., Scott F.H., Srisawat C., Engelke D.R. RNA affinity tags for the rapid purification and investigation of RNAs and RNA-protein complexes. Methods Mol. Biol. 2008;488:23–40. doi: 10.1007/978-1-60327-475-3_3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Johnson R.F., McCarthy S.E., Godlewski P.J., Harty R.N. Ebola virus VP35-VP40 interaction is sufficient for packaging 3E-5E minigenome RNA into virus-like particles. J. Virol. 2006;80:5135–5144. doi: 10.1128/JVI.01857-05. [DOI] [PMC free article] [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., Lamond A. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell Biol. 2008;183:223–239. doi: 10.1083/jcb.200805092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Satoh M., Shaheen V.M., Kao P.N., Okano T., Shaw M., Yoshida H., Richards H.B., 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. 1999;274:34598–34604. doi: 10.1074/jbc.274.49.34598. [DOI] [PubMed] [Google Scholar]
  • 27.Saunders L.R., Perkins D.J., Balachandran S., Michaels R., Ford R., Mayeda A., 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. 2001;276:32300–32312. doi: 10.1074/jbc.M104207200. [DOI] [PubMed] [Google Scholar]
  • 28.Reichman T.W., Muniz L.C., 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. 2002;22:343–356. doi: 10.1128/MCB.22.1.343-356.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Remold-O’Donnell E., Nixon J.C., Rose R.M. Elastase inhibitor. characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils. J. Exp. Med. 1989;169:1071–1086. doi: 10.1084/jem.169.3.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Cooley J., Takayama T.K., Shapiro S.D., Schechter N.M., Remold-O’Donnell E. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry. 2001;40:15762–15770. doi: 10.1021/bi0113925. [DOI] [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., Remold-O’Donnell E. Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an ovserpin family gene. Gene. 1998;213:179–187. doi: 10.1016/S0378-1119(98)00189-9. [DOI] [PubMed] [Google Scholar]
  • 33.Bird C.H., Blink E.J., Hirst C.E., Buzza M.S., Steele P.M., Sun J., Jans D.A., 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. 2001;21:5396–5407. doi: 10.1128/MCB.21.16.5396-5407.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Popova E.Y., Claxton D.F., Lukasova E., Bird P.I., Grigoryev S.A. Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood. Exp. Hematol. 2006;34:453–462. doi: 10.1016/j.exphem.2006.01.003. [DOI] [PubMed] [Google Scholar]
  • 35.Missen M.A., Haylock D., Whitty G., Medcalf R.L., Coughlin P.B. Stage specific gene expression of serpins and their cognate proteases during myeloid differentiation. Br. J. Haematol. 2006;135:715–724. doi: 10.1111/j.1365-2141.2006.06360.x. [DOI] [PubMed] [Google Scholar]
  • 36.Kao P.N., Chen L., Brock G., Ng J., Kenny J., Smith A.J., Corthesy B. Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90. J. Biol. Chem. 1994;269:20691–20699. [PubMed] [Google Scholar]
  • 37.Corthesy B., 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. 1994;269:20682–20690. [PubMed] [Google Scholar]
  • 38.Marcoulatos P., Koussidis G., Mamuris Z., Velissariou V., 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. 1996;16:1035–1038. doi: 10.1089/jir.1996.16.1035. [DOI] [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., Mathews M.B. Nuclear factor 45 (NF45) is a regulatory subunit of complexes with NF90/110 involved in mitotic control. Mol. Cell. Biol. 2008;28:4629–4641. doi: 10.1128/MCB.00120-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Merrill M.K., 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. 2006;80:6936–6942. doi: 10.1128/JVI.00243-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shim J., Lim H., Yates J., Karin M. Nuclear export of NF90 is required for interleukin-2 mRNA stabilization. Mol. Cell. 2002;10:1331–1344. doi: 10.1016/S1097-2765(02)00730-X. [DOI] [PubMed] [Google Scholar]
  • 42.Kuwano Y., Kim H.H., Abdelmohsen K., Pullmann R., Jr, Martindale J.L., Yang X., Gorospe M. MKP-1 mRNA stabilization and translational control by RNA-binding proteins HuR and NF90. Mol. Cell. Biol. 2008;28:4562–4575. doi: 10.1128/MCB.00165-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Xu Y.H., 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. 1999;68:441–454. doi: 10.1006/mgme.1999.2934. [DOI] [PubMed] [Google Scholar]
  • 44.Maruo N., Kobayashi Y., Horiuchi H., Kondo M., Fujita S. Histochemical study on the maturation of human megakaryocytes using microfluorometry. Histochemistry. 1992;97:141–145. doi: 10.1007/BF00267304. [DOI] [PubMed] [Google Scholar]
  • 45.Yanagida M., Shimamoto A., Nishikawa K., Furuichi Y., Isobe T., Takahashi N. Isolation and proteomic characterization of the major proteins of the nucleolin-binding ribonucleoprotein complexes. Proteomics. 2001;1:1390–1404. doi: 10.1002/1615-9861(200111)1:11<1390::AID-PROT1390>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  • 46.Chen C.Y., Gherzi R., Andersen J.S., Gaietta G., Jurchott K., Royer H.D., Mann M., Karin M. Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev. 2000;14:1236–1248. [PMC free article] [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., Vainchenker W. Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach. Blood. 2007;109:3225–3234. doi: 10.1182/blood-2006-07-037838. [DOI] [PubMed] [Google Scholar]

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