Functional investigation of the miRNA seed enrichments in the C5 group of intronic sequences. (A) Fourteen motifs enriched in the 270 retained introns of the C5 category identified by HOMER (Heinz et al. 2010). (B) Relative mRNA expression of RANBP9 and SRSF2 (predicted targets of miR-4716-5p) and TWF1 mRNA in HeLa cells transfected with mimics for miR-4716-5p (miR-4716 mimic) or a scrambled negative control (negative mimic). Values are normalized over GAPDH. Data are presented as mean ± SEM. t-test: (*) P < 0.05. Data points represent independent experimental repeats (n = 3). (C) Analysis of IR of C5 candidates ATP6V02 and NAA25 in HeLa cells transfected with mimics for miR-4716-5p or a scrambled negative control. Values are calculated as levels of IRT over total transcript expression for each candidate. Data are presented as mean ± SEM. t-test: (*) P < 0.05. Data points represent independent experimental repeats (n = 3). (D) Bar graphs showing qPCR analysis of DROSHA relative mRNA expression levels in MNs (DIV = 21) after being transfected with siRNA targeting DROSHA (DROSHAi) or a scrambled control (CTRL). Values are normalized over the geometric mean of housekeeping genes (GAPDH, POLR2B, UBE2D3) and expressed as fold change over mock transfected cells. Data are presented as mean ± SEM. Data are derived from three individual cell lines, one experimental block. Paired t-test: (***) P < 0.0005. (E) SDS-PAGE/western blotting showing expression levels of DROSHA and GAPDH in MNs (DIV = 21) transfected with siRNA targeting DROSHA, a scrambled control, or mock transfected cells (MOCK). GAPDH is assayed as a loading control. (F) Bar graphs showing qPCR analysis of candidate C5 IR levels in cytoplasmic (left) and whole-cell (right) MN (DIV = 21) samples upon DROSHA knockdown. IR ratio values are calculated as levels of IRT over total transcript expression for each candidate. Data are presented as mean ± SEM. Data are derived from three individual cell lines (whole cell) and two individual cell lines (cytoplasmic), one experimental block. Multiple paired t-tests with significance placed at <0.05. (ns) not significant.
