Fig. 1.

miRNAs target the initiation step of translation. HeLa cells were cotransfected with an R-luc mRNA, the F-luc control mRNA, and (where indicated) a synthetic miRNA. For each R-luc mRNA, transfections were performed with specific CXCR4 miRNA, with let-7 control miRNA, and without miRNA. R-luc activity from each transfection was normalized to the corresponding F-luc measurement. (A) The four R-luc test mRNAs. R-luc-4 sites and -0 sites are m⁷G(5′)ppp(5′)G-capped and polyadenylated and either contain or lack four target sites for miRNA CXCR4 (black squares). Two further variants of the R-luc-4 sites mRNA carried the wild-type CrPV intergenic IRES or an inactive mutant (indicated by the asterisks, ref. 37). The CrPV IRES mRNAs carry an A-cap and are not polyadenylated. (B) Repression by CXCR4 miRNA. Repression was calculated by dividing the normalized R-luc activity without miRNA by the normalized R-luc activity in the presence of miRNA. Bars represent averaged results mostly from three to five independent triplicate experiments (filled bars, CXCR4; open bars, let-7). Error bars indicate standard deviation where appropriate. (C) Activity of the CrPV IRES. Bars represent normalized R-luc activity with mutant or wild-type CrPV IRES mRNA (no miRNA added; expression from the wild-type IRES is expressed as 1.0). R-luc expression driven by the wild-type CrPV IRES was inefficient but still ≈3 orders of magnitude above background readings from mock-transfected controls. Averaged results from two independent triplicate experiments are shown.