LIN-28 binds endogenous let-7 genomic DNA. Synchronized PQ272 (LIN-28:GFP) or pD4792 (GFP) worms were collected at 10 h and analyzed by chromatin immunoprecipitation (ChIP) with polyclonal antibodies against RNA pol II, GFP or IgG. The ratio of the indicated genomic DNA in the immunoprecipitated sample to the input sample for at least three independent experiments is shown and was analyzed by Student's t-tests (*, p<0.05). Error bars show s.e.m.

Regulation of let-7 processing by lin-28. (a) Total RNA was isolated from WT N2 or lin-28(n719) embryos (E) and synchronized worms at the indicated time points, and analyzed by agarose and PAGE northern blotting. Representative blots from three independent experiments are shown. The arrowheads mark the location of the SL1 pri-let-7 transcript. (b) Total RNA was isolated and analyzed as in Figure 3a. Representative blots from three independent experiments are shown. (c-d) Levels of each pri-let-7 isoform at the 10 h time point in lin-28(n719) relative to WT N2 worms after 18s rRNA normalization were calculated from six independent northern blot experiments (c) or 3 independent qRT-PCR experiments (d) and analyzed by Student's t-tests (*, p<0.05**, p<0.005; ***, p<0.0005). Error bars show s.e.m.

Developmentally regulated processing of let-7 pri-miRNA transcripts. (a) Depiction of expected Drosha cleavage products: 5′ flanking, let-7 hairpin precursor, and 3′ flanking. The number of sequenced RACE clones that mapped to the precise 3′ and 5′ Drosha cleavage products at each time point from two independent experiments is shown. The sequences of all Drosha cleavage products are shown in Supplementary Figure 3. (b) RT-PCR was performed on two independent 5′ RACE samples from N2 (left panel) or N2 and lin-28(n719) worms (right panel). (c) Total RNA was isolated from synchronized eri-1(mg366) RNAi hypersensitive worms at the indicated time points after vector control (-) or pup-2 (+) RNAi treatment, and analyzed by agarose and PAGE northern blotting. Representative blots from three independent experiments are shown.

Expression of let-7 is transcriptionally and post-transcriptionally regulated. (a) Depiction of the 2460 nt long let-7 rescue construct with the positions of the mature let-7 sequence (shaded blue), 3′ splice site (SS; shaded yellow), two start sites (A and B) and approximate sizes of the spliced and unspliced transcripts indicated4,7. (b) Total RNA was isolated from embryos (E) or synchronized plet-7_B∷GFP transgenic worms and analyzed by northern blotting. The similar sized B and SL1 transcripts often do not clearly resolve. (c) Total RNA was isolated from embryos (E) or synchronized WT N2 worms and analyzed by agarose or PAGE northern blotting. Representative blots from four independent experiments are shown. (d) Average pri-, pre- and mature let-7 levels after normalization to 18s or 5.8s rRNA from four independent experiments. (e) Total RNA was isolated from synchronized plet-7_B∷GFP transgenic worms and analyzed as in Figure 1b. The entire blot is shown in Supplementary Figure 1c.

LIN-28 binds endogenous let-7 primary transcripts in C. elegans and human ES cells. (a-b) Total protein was isolated from PQ272 (LIN-28:GFP) worms and analyzed by western blotting. (b) Ratios of LIN-28:GFP levels to the 10 h time point after tubulin normalization were calculated from three independent experiments and analyzed by Student's t-tests (***, p<0.0005). Error bars show s.e.m. (c) Synchronized PQ272 worms were collected at 10 h and analyzed by RNA immunoprecipitation (RIP). Input, and LIN-28:GFP and IgG immunoprecipitates were analyzed by western blotting or RT-PCR. (d) Undifferentiated HUES6 cells were analyzed by RIP. Input, Lin28 and IgG immunoprecipitates were analyzed by western blotting or RT-PCR. (e) The worm and human cell samples from Figure 4c-d were analyzed by qRT-PCR to determine the levels of input or LIN-28 immunoprecipitated pri- or pre-let-7 RNAs using primers specific for pri-let-7 (pri_F and pri_R) or pre-let-7 and pri-let-7 transcripts containing the precursor hairpin (pre_F and pre_R). The ratio of precursor containing let-7 transcripts to pri-let-7 transcripts for immunoprecipitated samples after normalization to input samples for at least three independent experiments is shown, and was analyzed by Student's t-tests (*, p<0.05; **, p<0.005; ***, p<0.0005). Error bars show s.e.m. (f-g) Undifferentiated HUES6 cells were fractionationated into nuclear and cytoplasmic extracts before analysis by RIP and western blotting (f) or qRT-PCR as in Figure 4e (g). Results from three independent experiments are shown.