a, RT-PCR for pri-let-7g transcript (as described in ref. (7)) during ES differentiation to embryoid bodies. Actin serves as control. b, Northern blot showing post-transcriptional induction of mature let-7g during embryoid body formation 5S rRNA serves as loading control. c, in vitro pri-miRNA processing reaction using radiolabeled pri-let-7g as substrate. Pri-miRNA was pre-incubated with various amounts of P19 cell extract or mouse embryonic fibroblast (MEF) extract prior to processing reaction with Flag-Drosha immunoprecipitate, as described in Methods. The ratio of pre-miRNA to pri-miRNA was quantitated by densitometry and values were normalized to the Microprocessor only lane. d, qPCR analysis of gene expression during embryoid body formation of a feeder-free mouse ES line (J1 ES) . Top Panel: Pri-let-7g and mature let-7g; Middle Panel: Lin-28; Bottom Panel: pluripotency factors Oct-4 and Nanog.

a, α–Flag-Western to confirm expression of Flag-tagged proteins for use in in vitro assays. b, in vitro pri-miRNA processing reaction on pri-let-7g (left panel) and pri-let-7a (right panel) substrates in the presence of either Mock, Flag-Lin-28, Flag-hnRNPA1, or Flag-Msi-2 immunoprecipitate. Quantitation was normalized to the Microprocessor-only lane. c, in vitro pri-miRNA processing reaction on pri-let-7g (left panel) and pri-miR-15a/16-1 (right panel) substrates in the presence of either mock or Flag-Lin-28 immunoprecipitate and competitor tRNA. Quantitation was normalized to the Mock-IP lane. d, in vitro pri-miRNA processing reaction on pri-let-7g (left panel) and pri-let-7a (right panel) substrates in the presence of rHis-Lin-28. Quantitation was normalized to the Microprocessor-only lane.

a, In each panel, 293T cells were either untransfected (lane 1), co-transfected with the indicated pri-miRNA and 0.5 μg pCMV-Flag empty vector (lane 2), or co-transfected with the indicated pri-miRNA and 0.5 μg Flag-Lin-28 cDNA (lane 3). Total RNA was collected 40 h post-transfection and Northern blotted for the indicated miRNA. b, qPCR analysis of pri-let-7g levels for sample in a) (top right panel). c, Mature let-7g levels upon co-transfection of 293T cells with pri-let-7g and either pCMV-Flag, Flag-Lin-28, Flag-hnRNPA1, Flag-hnRNPL, Flag-YBX-1, or Flag-Msi-2 cDNAs, as measured by quantitative PCR. First, the amount of mature let-7g in each sample was calculated relative to untransfected control cells, then Flag-protein co-transfected samples were normalized to the corresponding pCMV-Flag co-transfected samples. d, qPCR showing changes in levels of endogenous mature miRNAs upon transfection of Flag-Lin-28 in 293T cells. e, qPCR showing accumulation of endogenous pri-let-7g upon transfection of Flag-Lin-28 in 293T cells. For c-e, values are given as average +/- S.E.M. from two or more independent transfections.

P19 cells were transfected with control hairpin (GFPi), pLKO.1-shRNA hairpins targeting Lin-28, control siRNA (scrambled sequence), or Lin-28 siRNA. Total RNA was collected 60-hrs post-transfection for analysis. a, quantitative PCR analysis of Lin-28 expression, normalized to Lin-28 expression with control hairpin or control siRNA, for samples in b. b, Northern blot for mature let-7g. c, confirmation of Lin-28 knockdown using Lin28-SI2 on samples analyzed in d. Error bars represent S.E.M. with N=3. d, Changes in mature miRNA levels upon knockdown of Lin-28 as analyzed by quantitative PCR. Error bars represent S.E.M. with N=3. e, levels of pri-let-7g upon knockdown of Lin-28 in P19 cells. Error bars represent S.E.M. with N=3. f, levels of the pluripotency markers Oct-4 and Nanog in P19 cells transfected with either control siRNA or Lin28-SI2. Error bars represent S.E.M. with N=3.