New features of micro-RNA regulation of mRNA translation and stability revealed by expression of targeted or not targeted reporter genes

Abstract

The existence of translation regulation by RNA-induced silencing complexes (RISCs) composed from Argonaute proteins and micro-RNAs is well established, however the mechanisms underlying specific cellular and miRNA effects and the way in which specific complexes arise are not completely clear. Here we describe experiments with Renilla and Firefly luciferase reporter genes transfected on a PsiCheck2 plasmid into human cancer HCT116 or Me45 cells where only the Renilla gene contained or not sequences targeted by micro RNAs (miRNAs) in the 3UTR. The effects of targeting were miRNA-specific; miRNA-21 caused strong inhibition of translation whereas miRNA-24 or Let-7 caused no change or an increase in global reporter Renilla luciferase synthesis, and the mRNA-protein complexes formed by reporter transcripts in both cell types differed as shown by sucrose gradient sedimentation. In both cell types the presence of miRNA targets on Renilla transcripts affected expression of the co-transfected non-targeted Firefly luciferase, and Renilla and Firefly transcripts were found in the same sucrose gradient fractions. We also observed that specific anti-miRNA oligoribonucleotides influenced expression of the Firefly as well as of the Renilla gene, suggesting modulation of non-targeted transcript expression by miRNAs. Our results indicate the existence of interactions between miRNA-regulated and -unregulated transcripts and suggest that the use of the latter as a normalizers in experiments may be biased. We also discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.