Selective blockade of microRNA processing by Lin28 - PubMed (original) (raw)

Selective blockade of microRNA processing by Lin28

Srinivas R Viswanathan et al. Science. 2008.

Abstract

MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked posttranscriptionally in embryonic stem cells, embryonal carcinoma cells, and primary tumors. Here we show that Lin28, a developmentally regulated RNA binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we found that Lin28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin28 as a negative regulator of miRNA biogenesis and suggest that Lin28 may play a central role in blocking miRNA-mediated differentiation in stem cells and in certain cancers.

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Figures

Figure 1

Figure 1. Post-transcriptional control of pri-let-7g processing

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.

Figure 2

Figure 2. Lin-28 Inhibits pri-miRNA processing in vitro

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.

Figure 3

Figure 3. Ectopic expression of Lin-28 selectively inhibits pri-miRNA processing in vivo

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.

Figure 4

Figure 4. Knockdown of Lin-28 relieves the miRNA-processing block

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.

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