Figure 4.

The upstream AUG triplets act as negative cis regulatory elements. (A). Schematic representation of reporter constructs with wild-type and mutant M2 5′-UTRs. The solid and open boxes indicate the upstream AUGs and luciferase genes, respectively. A30 represents the poly(A) tail of the in vitro RNA transcripts generated from these constructs. The free energy of the 5′-UTR in each construct was predicated using the mfold 3.1 algorithm (http://www.bioinfo.rpi.edu/~zukerm/rna/). In the mutants, the upstream AUGs were mutated to UUCs. (B) RNA transcripts of luciferase with different M2 5′-UTRs. The RNAs were generated from linearized reporter expression constructs by in vitro transcription using T7 RNA polymerase. (C) Translation of luciferase reporter with different M2 5′-UTRs. The translation of luciferase reporter was measured by the determination of the relative luciferase activity in HeLa cells following transfection of the RNA transcripts shown in (B) and normalization to the activity of the co-transfected β-galactosidase. (D) Real-time PCR analysis of RNA levels of luciferase reporter with different M2 5′-UTRs. In vitro transcripts of luciferase reporters with different M2 5′-UTRs were co-transfected into HeLa cells with transcripts of β-galactosidase and then real-time PCR analysis was performed as described in Figure 3 legend.