FIG 6.

DTMUV 5′ and 3′ UTR sequences and RNA secondary-structural elements are crucial for cap-independent translation. (A) Schematic presentation of the reporters used in this study. The diagrams are not to scale. Detailed information regarding the introduced mutations is presented in Table 1. (a) Predicted secondary structures in the UTRs of the DTMUV-RLuc reporter. UARs (i.e., upstream of AUG) and the DB pseudoknot are in purple. (b) Schematic presentation of reporter mRNAs harboring a deletion at the 5′ end or deletions of predicted secondary-structural elements. (c) Predicted native (wild-type [WT]) secondary structures of elements located in the 5′ UTR/3′ UTR and structures resulting from introduced mutations. Introduced substitutions are shown in red. (B) Translation efficiencies of DTMUV-RLuc reporters containing mutations shown in panel A. BHK-21 cells were cotransfected with the indicated uncapped reporters and uncapped huGL-FLuc control. The cells were lysed at 4 h p.t., and the activities of RLuc and FLuc were measured. The activities of RLuc expressed by different reporters were normalized to the activity of FLuc expressed by the transfection control. The relative luciferase activities (RLuc/FLuc) are presented as means plus SD (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s t test).