Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin.
Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.
Yu
Hong
H
Department of Medicine, Monash University, Box Hill Hospital, Victoria, Australia.
Stasinopoulos
Stan
S
Leedman
Peter
P
Medcalf
Robert L
RL
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005656:Fungal Proteins / Q000378:metabolism; D015723:Gene Library; D005914:Globins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000494:pharmacology*; D008957:Models, Genetic; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D051816:Tristetraprolin; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D005656:Fungal Proteins; D017874:Immediate-Early Proteins; D017396:Plasminogen Activator Inhibitor 2; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D051816:Tristetraprolin; C497552:ZFP36 protein, human; D005914:Globins