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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

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Correspondence to Odette Laneuville.

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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

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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