Retargeting of m169 to cellular and viral miRNAs.

<p><b>A</b>. Schematic representation of the miR-27a/m169 binding site and the introduced mutated binding sites resulting in retargeting of m169 to either mmu-miR-16 or mcmv-miR-M23-2. Target site predictions were performed using RNAhybrid to design the retargeting of m169. <b>B</b>. Efficient retargeting of m169 to a cellular and a viral miRNA. NIH-3T3 cells were infected with the indicated viruses and total RNA isolated at different times of infection was analyzed by northern blot for miR-27, miR-16 and mcmv-miR-M23-2. U6 served as loading control. Retargeting of m169 to miR-16 and mcmv-miR-M23-2 abolished degradation of miR-27 but resulted in highly efficient degradation of the respective miRNAs. <b>C</b>. Expression of m169 3′-UTR is sufficient to mediate miR-27 degradation. Adenoviral vectors expressing either full-length m169 or its 3′-UTR fused to EGFP were used to transduce NIH-3T3 cells at an MOI of either 50 or 500. An adenoviral vector expressing mCherry was used as negative control. Two days post-transduction, total RNA was prepared and miR-24/miR-27 ratios were determined by qRT-PCR.