Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3'-UTR.
Two cyclooxygenase (COX) enzymes, COX-1 and COX-2, are present in human cells. While COX-1 is constitutively expressed, COX-2 is inducible and up-regulated in response to many signals. Since increased transcriptional activity accounts for only part of COX-2 up-regulation, we chose to explore other RNA processing mechanisms in the regulation of this gene. Previously, we showed that COX-2 is regulated by alternative polyadenylation, and that the COX-2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs) that are very important in efficient polyadenylation. To explore trans-acting protein factors interacting with these cis-acting RNA elements, we performed pull-down assays with HeLa nuclear extract and biotinylated RNA oligonucleotides representing COX-2 USEs. We identified PSF, p54(nrb), PTB, and U1A as proteins specifically bound to the COX-2 USEs. We further explored their participation in polyadenylation using MS2 phage coat protein-MS2 RNA binding site tethering assays, and found that tethering any of these four proteins to the COX-2 USE mutant RNA can compensate for these cis-acting elements. Finally, we suggest that these proteins (p54(nrb), PTB, PSF, and U1A) may interact as a complex since immunoprecipitations of the transfected MS2 fusion proteins coprecipitate the other proteins.
Hall-Pogar
Tyra
T
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
Liang
Songchun
S
Hague
Lisa K
LK
Lutz
Carol S
CS
D020413:3' Untranslated Regions / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D050810:Octamer Transcription Factors / Q000378:metabolism; D000072670:PTB-Associated Splicing Factor; D026723:Polyadenylation; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism; D011485:Protein Binding; D039104:RNA 3' Polyadenylation Signals; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D013379:Substrate Specificity
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D038941:Polypyrimidine Tract-Binding Protein; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human