3'-Untranslated region of the type 2 bradykinin receptor is a potent regulator of gene expression.
Regulation of the constitutively expressed type 2 bradykinin (B2) receptor, which mediates the principal actions of bradykinin, occurs at multiple levels. The goal of the current study was to determine whether the human B2 3'-untranslated region (UTR) has effects on gene expression, with particular focus on the variable number of tandem repeats (B2-VNTR) polymorphic portion of the 3'-UTR and its flanking AU-rich elements (AREs). When inserted downstream of the luciferase coding region of the pGL3-Promoter vector, the B2-VNTR reduced reporter gene activity by 85% compared with pGL3-Promoter alone (promoter control; P < 0.001), an effect that was not appreciably affected by mutation of the flanking AREs. The negative regulatory effects of the B2-VNTR region were position and orientation dependent and strongly positively correlated with the number of tandem repeats in the B2-VNTR region (r = 0.85, P < 0.001). With respect to mechanism, quantitative RT-PCR revealed that the B2-VNTR mRNA level was 32% of that of promoter control (P = 0.008), whereas the number of polyadenylated transcripts was 4% (P = 0.02). In contrast, the mRNA half-life of the B2-VNTR was increased (B2-VNTR: 14.9 vs. promoter control: 12.2 h, P = 0.009). Transient transfection of human kidney-derived tsA201 cells with the B2-VNTR construct increased transcription of the native B2 receptor mRNA by 43% (P < 0.05), supporting an endogenous B2 receptor-regulatory capacity of the B2-VNTR. In conclusion, these results identify novel pretranslational effects of the B2-VNTR region to act as a potent negative regulator of heterologous gene expression and support the notion that the bradykinin B2 3'-UTR may impact endogenous receptor regulation.
Zamorano
Rocio
R
Vanderbilt University Medical Center, Nashville, TN 37232-6602, USA.
Suchindran
Sunil
S
Gainer
James V
JV
D020413:3' Untranslated Regions; D001483:Base Sequence; D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D026723:Polyadenylation; D012333:RNA, Messenger / Q000096:biosynthesis; D043782:Receptor, Bradykinin B2 / Q000235:genetics* / Q000502:physiology*; D017385:Sequence Homology; D014162:Transfection
D020413:3' Untranslated Regions; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D043782:Receptor, Bradykinin B2; D003609:Dactinomycin