Comparison of activities of various 5′UTRs in monocistronic versus dicistronic contexts. (A) Schematic representation of mono- and dicistronic mRNAs used in the assay. (B and C) HEK293 and NTera2/D1 cells were transfected with either dicistronic mRNA derivatives as indicated or with an equimolar mixture of the corresponding monocistronic reporter (coding for Fluc) and the normalizing mRNA (Rluc). Fluc/Rluc values for monocistronic mRNA were taken as 100 units for each individual construct. (D) Results of mono- and dicistronic mRNA translation in in vitro “RRL+HeLa” cell-free system. Lane 1, Rluc; lane 2, L1 5′UTR-Fluc; lane 3, equimolar mixture of L1 5′UTR-Fluc and Rluc; lane 4, dicistronic Rluc-L1 5′UTR-Fluc; lane 5, mixture of L1 5′UTRΔ(133-887)-Fluc and Rluc; lane 6, dicistronic Rluc-L1 5′UTRΔ(133-887)-Fluc.