title: Identification of tristetraprolin as a factor that modulates the stability of the TAFI transcript through binding to the 3'-untranslated region.
abstract: BACKGROUND
  Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen encoded by the human gene CPB2. TAFI constitutes a molecular link between coagulation and fibrinolysis, and between coagulation and inflammation. The 3'-untranslated region (UTR) of the human CPB2 mRNA plays a key role in regulating CPB2 mRNA abundance, but the exact mechanisms that mediate this regulation are largely unexplored.
  
  
  OBJECTIVE
  To pinpoint cis-acting elements in the CPB2 3'-UTR that act as stability determinants and to identify protein factors binding to these sites.
  
  
  METHODS
  We constructed a series of plasmids encoding mRNAs containing rabbit β-globin sequences (as a reporter) fused to sequences of the CPB2 3'-UTR (encompassing 5' and internal deletions). These plasmids were transfected into HepG2 (human hepatoma) cells and the stability of the fusion transcripts measured. We performed a series of gel mobility shift analyses using RNA probes encompassing putative (in)stability elements.
  
  
  RESULTS
  We identified one element conferring stability and three elements conferring instability. Supershift assays identified the protein bound to the site between the second and third polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP site abolished TTP binding in gel mobility shift assays and also stabilized β-globin/CPB2 fusion transcripts. TTP knockdown stabilized the fusion transcript containing the TTP site, but not a fusion transcript in which this site was mutated.
  
  
  CONCLUSIONS
  Our findings are indicative of a role for TTP in constitutive, and perhaps regulated, control of CPB2 mRNA stability and hence abundance.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Novakovic
authors.forename: A C Y
authors.initials: AC
authors.lastname: Kuo
authors.forename: J H
authors.initials: JH
authors.lastname: Lin
authors.forename: M L
authors.initials: ML
authors.lastname: Koschinsky
authors.forename: M B
authors.initials: MB
authors.lastname: Boffa
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001609:Beta-Globulins / Q000235:genetics; D001665:Binding Sites; D025901:Carboxypeptidase B2 / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D056945:Hep G2 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D051816:Tristetraprolin / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D001609:Beta-Globulins; D012333:RNA, Messenger; D051816:Tristetraprolin; C497552:ZFP36 protein, human; D025901:Carboxypeptidase B2