Identification of RNA-binding Proteins in RAW 264.7 Cells That Recognize a Lipopolysaccharide-responsive Element in the 3-Untranslated Region of the Murine Cyclooxygenase-2 mRNA

摘要

RAW 264.7 cells rapidly induce cyclooxygenase-2 (COX-2) in response to lipopolysaccharide treatment. Part of the increased COX-2 expression occurred through post-transcriptional mechanisms mediated through specific regions of the 3′-untranslated region (UTR) of the message. The proximal region of the 3′-UTR of COX-2 contains a highly conserved AU-rich element that was able to confer lipopolysaccharide regulation of a chimeric reporter-gene. Electrophoretic mobility shift assays demonstrated that the RNA-binding proteins TIAR, AUF1, HuR, and TIA-1 all form an RNA-protein complex with the first 60 nucleotides of the 3′-UTR of COX-2. Biotinylated RNA probes were used to isolate additional proteins that bind the 3′-UTR of COX-2. We identified several RNA-binding proteins including TIAR, AUF1, CBF-A, RBM3, heterogeneous nuclear ribonucleoprotein (hnRNP) A3, and hnRNP A2/B1. We identified four alternatively spliced isoforms of AUF1 which migrated at multiple isoelectric points. Likewise, we identified alternatively spliced isoforms of CBF-A, hnRNP A3, and hnRNP A2/B1. Western analysis of two-dimensional gels identified multiple isoforms of TIA-1, TIAR, and AUF1 at pI values that spanned nearly 3 pH units. Thus, through a combination of alternative splicing and post-translational modification cells are able to increase greatly the repertoire of protein species expressed at a given time or in response to extracellular stimuli. RAW 264.7 cells rapidly induce cyclooxygenase-2 (COX-2) in response to lipopolysaccharide treatment. Part of the increased COX-2 expression occurred through post-transcriptional mechanisms mediated through specific regions of the 3′-untranslated region (UTR) of the message. The proximal region of the 3′-UTR of COX-2 contains a highly conserved AU-rich element that was able to confer lipopolysaccharide regulation of a chimeric reporter-gene. Electrophoretic mobility shift assays demonstrated that the RNA-binding proteins TIAR, AUF1, HuR, and TIA-1 all form an RNA-protein complex with the first 60 nucleotides of the 3′-UTR of COX-2. Biotinylated RNA probes were used to isolate additional proteins that bind the 3′-UTR of COX-2. We identified several RNA-binding proteins including TIAR, AUF1, CBF-A, RBM3, heterogeneous nuclear ribonucleoprotein (hnRNP) A3, and hnRNP A2/B1. We identified four alternatively spliced isoforms of AUF1 which migrated at multiple isoelectric points. Likewise, we identified alternatively spliced isoforms of CBF-A, hnRNP A3, and hnRNP A2/B1. Western analysis of two-dimensional gels identified multiple isoforms of TIA-1, TIAR, and AUF1 at pI values that spanned nearly 3 pH units. Thus, through a combination of alternative splicing and post-translational modification cells are able to increase greatly the repertoire of protein species expressed at a given time or in response to extracellular stimuli. Macrophages play a pivotal role in potentiating the proinflammatory response. Activation of macrophages with bacterial lipopolysaccharides (LPS) 1The abbreviations used are: LPS, lipopolysaccharide(s); ARE, adenylate/uridylate-rich element; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; hnRNP, heterogeneous nuclear ribonucleoprotein; MALDI, matrix-assisted laser desorption/ionization; UTR, untranslated region. 1The abbreviations used are: LPS, lipopolysaccharide(s); ARE, adenylate/uridylate-rich element; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; hnRNP, heterogeneous nuclear ribonucleoprotein; MALDI, matrix-assisted laser desorption/ionization; UTR, untranslated region. leads to production and secretion of various cytokines and prostaglandins. Prostaglandin production requires induction of cyclooxygenase-2 (COX-2), which catalyzes the conversion of arachidonic acid to pro