Fig. 6

Structure in the 5′UTR of MCL-1 mRNA affects its translation efficiency. a The minimal-free energy (MFE) structure for the 80-nucleotide MCL-1 5′UTR and the first 21 nucleotides of its coding region, as assigned by the Vienna RNA Websuite. The optimal MFE structure shown is −26.4 kcal/mol, while the free energy of the thermodynamic ensemble of predicted structures is −28.55 kcal/mol. b In the dual reporter vector pMCL-1 5′UTR, the 5′UTR of the CMV major immediate early region mRNAs, between the CMV promoter and the renilla luciferase coding region, is replaced by the full 80 bp of the MCL-1 5′ UTR plus the first 13 nucleotides of the MCL-1 coding region, since the latter contributes to the predicted secondary structure in a. c Efficiency of the MCL-1 5′UTR compared with that of CMV in producing renilla luciferase and firefly luciferase as a control, in the absence of 2DG. NALM-6 cells were electroporated with the two reporter constructs and the levels of the two luciferases determined 6 h later (see Fig. 5b and Methods). The MCL-1 5′UTR drove renilla luciferase only half as efficiently as the CMV 5′UTR (n = 3), whether the raw data was compared (left panel), or the renilla data was normalized to that obtained with firefly luciferase (right panel), to preclude any differences due to electroporation efficiency or transcription. In fact, the firefly results for the two constructs did not differ significantly. d Reduction by 2DG in translation of renilla luciferase driven by the MCL-1 5′UTR. As in c, NALM-6 cells electroporated with the MCL-1 5′UTR were treated with 1, 5, or 10 mM 2DG for 6 h and the two luciferases then assayed (see Methods). The % reductions in translation by 2DG (n = 3) are comparable to those with the CMV 5′UTR (Fig. 5b) but highly significant even with 1 mM 2DG