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.
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.
Cok
Steven J
SJ
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Acton
Stephen J
SJ
Sexton
Alison E
AE
Morrison
Aubrey R
AR
D020413:3' Untranslated Regions / Q000235:genetics; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D017124:Conserved Sequence; D051546:Cyclooxygenase 2; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; C000630987:Hnrpd protein, mouse; D007527:Isoenzymes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C489665:Tial1 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases