Translation of reporter mRNAs is repressed by endogenous let-7a in HeLa cells. (A) The sequence of the C. elegans lin-41 3′ UTR is shown with the two experimentally confirmed (33) LCSs in boxes. The 85-bp sequence containing two LCSs and a spacer region that was inserted into reporter constructs is shown in bold. (B) The plasmid constructs used in C–F. FF4LCS contains the FF luciferase ORF with two of the boldfaced regions in A, containing four boxed LCSs, inserted into the 3′ UTR. FFr4mLCS contains the FF luciferase reporter with the bolded region in reverse orientation and the four LCSs mutated. Each LCS is shown as a white box, and mutated LCSs are gray boxes, with arrows denoting forward or reverse orientation. The FF reporters were transcribed from the CMV or the HSV-TK promoter. Also shown are the cotransfected R luciferase reporter under the control of the alternate promoter and the equation used to calculate the translation efficiency. (C) HeLa cells were transfected with plasmids depicted in B using the cationic lipid formulation TransIT-HeLaMONSTER (Mirus). FF luciferase activity was measured and normalized to R luciferase activity, and then the forward LCS-containing constructs were normalized to the reversed, mutated control (FF4LCS/FFr4mLCS; FFr4mLCS value set to 1). The first lane represents the untransfected control, which had no activity above background. (D) Denaturing, polyacrylamide gel analysis of RNA harvested from transfected cells and subjected to RNase protection analysis. (E) The gel of the RNase protection analysis in D was quantified, and the FF mRNA value was normalized to R mRNA, which was then normalized to the reversed, mutated control (value set to 1). (F) The luciferase activity in C was normalized to the mRNA levels determined in E and then normalized again to their reversed, mutated controls to obtain the translation efficiency. Error bars represent standard deviations from three experiments.

HCV IRES-containing reporter mRNAs are repressed by LCSs in the 5′ UTR. (A) The HCV IRES was inserted into the 5′ UTR of the FF luciferase reporter with the first 40 aa of the HCV core protein fused to the N terminus of the FF luciferase. A 54-bp hairpin (hp) (38, 39) was added upstream of the IRES to prevent leaky scanning of ribosomes from the mRNA cap and allow translation only from the internal ribosome entry codon. Four LCSs (4LCS; shown as white boxes) were inserted into the 5′ UTR before or after the hairpin or into the 3′ UTR of the reporters. Arrows depict the forward or reverse orientation of the LCSs. (B) HeLa cells were transfected as in Fig. 1 with the plasmid constructs depicted in A. FF luciferase activity was measured and normalized as described in Fig. 1C. (C–E) Translation efficiency was analyzed and calculated as described in Fig. 1 C–F. Error bars represent standard deviations from five experiments.

The degree of translational repression differs when DNA versus RNA is transfected. (A–C) HeLa cells were transfected with the plasmid constructs depicted in Figs. 1B and 2A using the cationic lipid transfection reagent, Lipofectamine 2000 (Invitrogen). The results were analyzed as in Figs. 1 and 2. (D–F) HeLa cells were transfected using Lipofectamine 2000 with RNAs in vitro-transcribed from the plasmids described in A. The mRNAs were G-capped and polyadenylated. FF luciferase activity was measured and normalized as in A–C. Error bars represent standard deviations from at least three experiments. (G–I) HeLa cells were transfected, and the results were normalized as in D–F except that the mRNAs were A-capped and polyadenylated. The results for FF4LCS and its reverse control are not shown because all values were below detectable limits. Bars represent the average of two experiments.

Electroporation yields similar conclusions for DNA versus RNA transfections. (A–C) HeLa cells were transfected with the plasmid constructs depicted in Figs. 1B and 2A by electroporation. FF luciferase activity was assayed, and translation efficiency was calculated as in Fig. 1 C–F. Error bars represent standard deviations from three experiments. (D–F) HeLa cells were transfected by electroporation with G-capped, polyadenylated mRNAs in vitro-transcribed from the plasmids described in A and assayed as above. Error bars represent standard deviations from three experiments.

Translation is repressed when human Ago2 is tethered to either the 5′ or 3′ UTR of IRES-containing reporters. (A) The hAgo2 protein, with theλN and HA domains fused to its N terminus, or the same protein with the first 62 aa of Ago2 deleted (ΔPRP), was coexpressed with IRES-containing FF reporter RNAs. The reporters contained five boxB (5BB) N-protein-binding sites located 5′ to the hairpin (hp) upstream of the HCV IRES in the 5′ UTR or in the 3′ UTR. Control constructs contained five reversed boxB sequences (r5BB). (B) HeLa cells were transfected by using TransIT-HeLaMONSTER (Mirus) with the plasmids described in A, and, after normalization of the luciferase activity to the mRNA levels as in Fig. 1F, the forward boxB constructs were normalized to the reversed boxB constructs and the tethered Ago2 results were normalized to the Ago2ΔPRP results (value set to 1). Error bars represent standard deviations from three to four experiments. Similar results were obtained with normalization to values obtained with no coexpression of the N fusion protein.