miR-101 and 5’-isomiR-101 bind Ago2 and Rck/p54. A. Specificity of miR-101 and 5’-isomiR-101 detection. SH-SY5Y cells were transfected with a negative control sequence (siGLO Green) or the mIRIDIAN mimics for miR-101 (dark bars) or 5’-isomiR-101 (light bars). Specific RT-qPCRs determinations for miR-101 and 5’-isomiR-101 were performed. Results are expressed as the Fold Change ratios (miridian mimic / control siGLO). B. Expression of exogenously transfected miR-101 or 5’-isomiR-101 in Ago2 immunocomplexes. SH-SY5Y cells were transfected with Siglo Green or the mIRIDIAN mimics for miR-101 or 5’-isomiR-101. Specific RT-qPCRs were performed in Ago2-FLAG immunoprecipitates.Results are expressed as the Fold Change ratios obtained from the FLAG antibody/Control antibody immunoprecipitates. Western-blot with anti-FLAG antibody shows the presence of Ago2-FLAG in the FLAG-IP and input, and the absence of Ago2-FLAG in the control IP. C. Detection of RISC components in miR-101 or 5’-isomiR-101 pull-down. SH-SY5Y cells stably expressing Ago2-FLAG were transfected with mIRIDIAN 3’-biotin-miR-101 or 3’-biotin-5’-isomiR-101. Biotin-miRNA complexes were pulled down with streptavidin beads and subsequently assayed for western blot analysis using Ago2 and Rck/p54 antibodies. The relative amount of Ago2 and Rck/p54 in 3’-biotin-5’-isomiR-101 versus 3’-biotin-miR-101 is shown in the middle panel. miRNA-101 transfected samples were used to normalize data and were assigned a value of 1. Left panel shows similar amounts of transfected 3’-biotin-miR-101 or 3’-biotin-5’-isomiR-101 determined in total cell extracts with the corresponding specific RT-qPCR assays. D. Expression of endogenous miR-101 and 5’isomiR-101 in Ago2-immunocomplexes in the human brain. Ago2 complexes were immunoprecipitated from human frontal cortex homogenates and miR-101, 5’-isomiR-101, miR-29a, U6 SNORD44 were determined by RT-qPCR. Results are expressed as the Fold Change ratios obtained from the Ago2 vs denatured Ago2 immunoprecipitates. Three independent experiments were performed in A- D. All data are expressed as the mean± SEM. Asterisks indicate statistical significance between miR-101 and 5’-isomiR-101 data : * (p ≤ 0.05), ** (p≤0.01) using Mann–Whitney test.