Figure 2

The AID 3'UTR is recognized by miRs -93 and -155. (A) Diagrams of miR and siRNA expression vectors. 5'Alu corresponds to a genomic fragment containing the miR-517a upstream Alu promoter [32]. In pAL-1, shLacZ replaces the miR-517a hairpin. In pALs-155 and -93, the pre-miR-155 and -93 hairpins replace the miR-517a hairpin respectively. TTTT, RNA polymerase III termination signal. (B) Diagrams of luciferase reporters. The AID 3'UTR luciferase reporter (AID 3'LR) and control 3'UTR luciferase reporter (Ctl 3'LR) were generated respectively by placing an AID 3'UTR sequence containing both the miR-155 and miR-93 target sites and a specific region of LacZ previously shown to be targeted by the pAL-1 shLacZ hairpin in a multiple cloning site in the Renilla luciferase 3'UTR [32]. HSV-TKp, herpes simplex virus thymidine kinase promoter; SV40p, simian virus 40 promoter; FF Luc, firefly luciferase; Ren Luc, Renilla luciferase; pA, poly(A). (C) AID 3'LR is specifically repressed by both miR-93 and miR-155 in 293 transient transfections. Luciferase assays (n = 4) of HEK293 lysates after transfection of AID 3'LR and either pAL-1, pAL-155 or pAL-93. Transfections were normalized to AID 3'LR alone. RLU, relative light units. (D) Ctl 3'LR is specifically repressed by the ctl (pAL-1) in 293 transient transfections. Cotransfections done as in (C), except Ctl 3'LR replaced AID 3'LR.