Fig. 5
miR-346 Induces DNA Damage in part Independently of NORAD/PUM2, NORAD Promotes Target-Directed miR-346 Decay. A Western blot analysis of phospho-Ser139-γH2AX protein levels in LNCaP cells transfected with 20 nM miR-346 for indicated duration. β-actin was used as a loading control. B, C qRT-PCR analysis of B) NORAD and C) miR-346 in cytosolic, soluble nuclear and chromatin fractions of 22RV1 cells. SNORD48 was used as a fractionation control and identified almost exclusively complexed with chromatin (Fig. S12B). D Immunofluorescent microscopy analysis of i,iii) phospho-Ser139-γH2AX protein levels in C42 cells transfected with miR-346 or NC (20 nM) ± siPUM2 (20 nM) for 72 h. E Illustration of NORAD:miR-346 putative interaction sites with features consistent with target-directed microRNA decay. F qRT-PCR quantification of number of miR-346 copies and NORAD TDMD sites in unperturbed C42B cells. Ten-fold serial dilutions of a miR-346 mimic and NORAD qPCR amplicon were prepared, reverse transcribed and analysed by qRT-PCR in parallel to C42B samples for absolute quantification. G qRT-PCR analysis of miR-346, − 221-3p and 222-3p levels in C42 cells transfected with siNORAD or siNC for 72 h. miR levels were normalised to U6, H qRT-PCR analysis of miR-346 levels in 22RV1 cells transfected with pcDNA3.1-NORAD WT or TDMD mutant for 72 h. miR levels were normalised to U6. Columns: mean ± SEM for three independent experiments performed in triplicate. J Western blot analysis of phospho-Ser139-γH2AX protein levels in C42 cells transfected with miR-346 (10 nM) ± WT or TDMD-mutant NORAD. β-actin was used as a loading control. A representative image of four independent experiments is shown. Densitometry was performed using ImageJ. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0001. See also Fig. S12, Table S7