Figure 3.

3p miRNAs derived from 5′-extended pre-miRNAs induce RNA silencing. (A) Lysates from 293T cells transfected with plasmids encoding different 5′-extended pre-miR-HSUR4 were subjected to Ago IP. Ago-enriched RNAs were analyzed by Northern blot using probes for miR-HSUR4-5p, -3p, endogenous miR-16, and U6. S: supernatant; P: pellet. In the upper panel, open triangles point to different pre-miR-HSUR4s, while the solid triangles point to miR-HSUR4-5p. The asterisk (*) marks degradation products observed during IP incubation (see also Supplementary Figure S6). Such products were not observed when total RNA was extracted by Trizol reagent directly (Figures 2B and 4A). (B) GFP reporter for miR-HSUR4-5p and (C) miR-HSUR4-3p were cotransfected with pcDNA3, pU1-HSUR4, or pU1-miR-HSUR4 with various 5′ extensions. pcDNA3 is an empty vector used as a negative control. pU1-HSUR4 was used as a positive control. GFP fluorescence and bright field images of the cells after 48 h are shown.