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Selective Blockade of MicroRNA Processing by Lin28

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Science  04 Apr 2008:
Vol. 320, Issue 5872, pp. 97-100
DOI: 10.1126/science.1154040

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.

MicroRNAs (miRNAs) constitute a large family of short, noncoding RNAs that posttranscriptionally repress gene expression in metazoans. Mature miRNAs are produced from primary miRNA transcripts (pri-miRNAs) through sequential cleavages by the Microprocessor, comprising the ribonuclease III Drosha component and the double-stranded RNA (dsRNA) binding protein DGCR8 (1, 2) and Dicer (3, 4) enzyme complexes, to release pre-miRNA and mature miRNA species, respectively. Posttranscriptional control of miRNA expression has been reported to occur in a tissue-specific (5) and developmentally regulated fashion (68). The processing of several pri-miRNAs is blocked in embryonic tissues, with activation of processing occurring only as development proceeds. In addition, it has been reported that certain pri-miRNAs are highly expressed in human (9) and mouse embryonic stem (ES) cells, mouse embryonal carcinoma (EC) cells, and human primary tumors; however, the corresponding mature species are not detectable (7). This suggests that there may be a posttranscriptional block in miRNA biogenesis, the mechanism of which has remained unknown. In ES and EC cells, the magnitude of the Microprocessor processing block is greatest for members of the let-7 family of miRNAs, although it has been proposed that the processing of all miRNAs may be regulated at the Microprocessor step (7).

We observed that the pri-let-7g transcript is readily detectable in ES cells and remains at relatively constant levels over the course of differentiation into embryoid bodies (Fig. 1A). In contrast, mature let-7g is undetectable in undifferentiated ES cells but is strongly induced after day 10 of differentiation (Fig. 1B). A posttranscriptional induction of let-7g expression has also been reported during the differentiation of P19 EC cells with retinoic acid (7). We sought to understand the mechanism for the posttranscriptional block in miRNA processing in EC and ES cells. We first compared cell extracts from different cell types for their ability to inhibit Microprocessor-mediated cleavage of pri-miRNA substrates to the corresponding pre-miRNAs in vitro (Fig. 1C). Radiolabeled pri-miRNA substrates were preincubated with cell extract and were then subjected to processing by affinity-purified Microprocessor complex. Whereas extracts from undifferentiated P19 cells readily inhibited Microprocessor-mediated cleavage of pri-let-7g to pre-let-7g, cell extracts from differentiated mouse embryonic fibroblasts (MEFs) did not inhibit cleavage. Thus, the cell-type specificity of the in vivo Microprocessor processing block was recapitulated in our in vitro assay.

Fig. 1.

Posttranscriptional control of pri-let-7g processing. (A) Reverse transcription PCR for pri-let-7g transcript [as described in (7)] during ES differentiation to embryoid bodies. Actin serves as control. (B) Northern blot showing posttranscriptional 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 preincubated with various amounts of P19 cell extract or mouse embryonic fibroblast (MEF) extract before processing reaction with Flag-Drosha immunoprecipitate (see supporting online material). The ratio of pre-miRNA to pri-miRNA was quantitated by densitometry and values were normalized to the Microprocessor-only lane. (D) Quantitative PCR analysis of gene expression during embryoid body formation of a feeder-free mouse ES line (J1 ES). Data are the average of experimental triplicates performed on the RNA samples from (B). Top, Pri-let-7g and mature let-7g; middle, Lin28; bottom, pluripotency factors Oct-4 and Nanog.

We surmised that a protein factor or factors present in ES and EC cells might be inhibiting Microprocessor-mediated processing of pri-miRNAs, and we used a biochemical approach to identify this factor. Electrophoretic gel mobility shift assays using a labeled pre-let-7g probe identified a specific band shift present in P19 EC and ES extract but not with MEF extract (fig. S1). This finding suggested that pre-let-7g could be used as an effective affinity reagent for purification of the factor(s) responsible for the Microprocessor processing block. Pre-let-7g was conjugated to agarose beads and incubated with whole-cell extract from P19 cells. The affinity eluate was subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by colloidal staining. Bands were excised and subjected to mass spectroscopic sequencing in three segments (fig. S2). Sequencing revealed several RNA binding proteins copurifying with pre-let-7g. A number of these proteins were previously identified as members of a large Drosna-containing protein complex (2) (fig. S2).

One of the pre-let-7g–interacting proteins was the small, highly conserved RNA binding protein Lin28. Lin28 was an attractive candidate for the following reasons: (i) Mutations within its RNA binding domain have been shown to impair developmental timing regulation in Caenorhabditis elegans (10); (ii) it is expressed specifically in undifferentiated P19 cells, mouse ES cells (11), and human ES cells (12) and is down-regulated upon differentiation; (iii) a mammalian Lin28 homolog, Lin28B, is overexpressed in hepatocellular carcinoma, and overexpression of this gene promotes cancer cell proliferation in vitro (13); (iv) it has been reported that Lin28 is expressed in embryonic muscle, neurons, and epithelia in a stage-specific fashion, and Lin28 is crucial for appropriate skeletal muscle differentiation (11); and (v) Lin28 was recently used with three other factors to reprogram human somatic fibroblasts to pluripotency (14).

We examined the kinetics of Lin28 expression during embryoid body formation (Fig. 1D). Lin28 is down-regulated upon ES cell differentiation, with kinetics that are delayed relative to the known pluripotency factors Oct-4 and Nanog. This down-regulation of Lin28 temporally coincides with activation of pri-let-7 processing (Fig. 1D).

To explore the possibility that Lin28 may regulate pri-let-7 processing, we confirmed that Lin28 is capable of binding both pre-let-7g and pri-let-7g in a cosedimentation assay (fig. S3). We then tested the ability of Lin28 to functionally block pri-miRNA processing in vitro. We observed that a Flag immunoprecipitate containing Flag-Lin28 potently inhibited the processing of both pri-let-7a and pri-let-7g in vitro. Flag immunoprecipitates containing the control RNA binding proteins Flag-hnRNPA1 and Flag–Msi-2 had no effect on pri-let-7a and pri-let-7g processing (Fig. 2, A and B). Flag-Lin28 immunoprecipitate did not impair the processing of pri-miR-15a/16-1, which demonstrates the selectivity of the miRNA processing block (Fig. 2C). We next purified bacterially expressed His-Lin28 (Fig. 2D) and tested this recombinant Lin28 (rLin28) for its ability to block pri-miRNA processing in vitro. rLin28 inhibited the processing of both pri-let-7a and pri-let-7g (Fig. 2D). Therefore, Lin28 is sufficient to inhibit miRNA processing at the Microprocessor step.

Fig. 2.

Lin28 inhibits pri-miRNA processing in vitro. (A) α-Flag–Western blot 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 mock, Flag-Lin28, 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-Lin28 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-Lin28. Quantitation was normalized to the Microprocessor-only lane.

To determine whether Lin28 is capable of blocking miRNA processing in vivo, we introduced four pri-miRNAs in either the presence or absence of mouse Lin28 cDNA into 293T cells, a transformed human cell line that lacks Lin28. In the absence of Lin28, all ectopic pri-miRNAs were efficiently processed to their mature form (Fig. 3A and fig. S4). However, ectopic expression of Lin28 completely blocked processing of both pri-let-7a and pri-let-7g, whereas processing of pri-miR-15a and pri-miR-122 was largely unaffected (Fig. 3A and fig. S4). Cotransfection of pri-let-7g and Lin28 led to accumulation of pri-let-7g (Fig. 3B), consistent with the notion that Lin28 blocks miRNA processing at the Microprocessor step. We performed these cotransfection experiments with four control RNA binding proteins (YBX-1, Msi-2, hnRNPA1, and hnRNPL) to confirm that this block in processing of pri-let-7 miRNAs is specific to Lin28 (Fig. 3C and fig. S5). Finally, to test whether Lin28 is capable of blocking endogenous miRNA processing (as opposed to only blocking the processing of ectopically expressed pri-miRNAs), we transfected Lin28 cDNA into 293T cells and measured levels of several mature miRNAs after 4 days by quantitative polymerase chain reaction (PCR). We observed decreased endogenous levels of mature let-7 family members; levels of endogenous mature miR-21 were unaffected (Fig. 3D). Decreased mature let-7g upon Lin28 overexpression was accompanied by a corresponding increase in levels of pri-let-7g (Fig. 3E).

Fig. 3.

Ectopic expression of Lin28 selectively inhibits pri-miRNA processing in vivo. (A) In each panel, 293T cells were untransfected (lane 1), cotransfected with the indicated pri-miRNA and 0.5 μg of pCMV-Flag empty vector (lane 2), or cotransfected with the indicated pri-miRNA and 0.5 μg of Flag-Lin28 cDNA (lane 3). Total RNA was collected 40 hours after transfection and subjected to Northern blotting for the indicated miRNA. (B) Quantitative PCR analysis of pri-let-7g levels [average of experimental triplicates performed on the RNA samples from upper right panel of (A)]. (C) Mature let-7g levels upon cotransfection of 293T cells with pri-let-7g and pCMV-Flag, Flag-Lin28, 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 cotransfected samples were normalized to the corresponding pCMV-Flag cotransfected samples. (D) Quantitative PCR showing changes in levels of endogenous mature miRNAs upon transfection of Flag-Lin28 in 293T cells. (E) Quantitative PCR showing accumulation of endogenous pri-let-7g upon transfection of Flag-Lin28 in 293T cells. For (C) to (E), values are given as average ± SEM from two or more independent transfections.

We sought to determine whether Lin28 is an endogenous blocker of miRNA processing in embryonic cells. We used three different short hairpin RNAs (shRNAs) and a small interfering RNA (siRNA) targeting Lin28 to knock down endogenous Lin28 in P19 EC cells (Fig. 4A) and ES cells (fig. S6). Knockdown of Lin28 led to an induction of mature let-7g in both P19 cells (Fig. 4B) and ES cells (fig. S4B), indicating that Lin28 serves to inhibit miRNA processing in vivo (Fig. 4B). All let-7 family members tested were substantially up-regulated upon knockdown of Lin28, whereas levels of other miRNAs were unchanged (Fig. 4D and fig. S7). Induction of mature let-7 miRNAs occurred within 60 hours of Lin28 knockdown, whereas let-7 miRNAs were normally induced only after 10 days of ES and P19 differentiation, when endogenous Lin28 levels fell (Fig. 1A) (7). Therefore, the induction we observed likely represents a direct effect of Lin28 on pri-miRNA processing rather than an indirect consequence of cell differentiation. In support of this notion, we observed no decrease in levels of the pluripotency markers Oct-4 and Nanog upon knockdown of Lin28 over the time course of our experiment (Fig. 4F). Furthermore, global miRNA profiling detected up-regulation of only let-7 miRNAs upon Lin28 knockdown, underscoring the specificity of Lin28 in regulating let-7 miRNAs (fig. S7).

Fig. 4.

Knockdown of Lin28 relieves the miRNA-processing block. P19 cells were transfected with control hairpin (GFPi), pLKO.1-shRNA hairpins targeting Lin28, control siRNA (scrambled sequence), or Lin28 siRNA. Total RNA was collected 60 hours after transfection for analysis. (A) Quantitative PCR analysis of Lin28 expression, normalized to Lin28 expression with control hairpin or control siRNA [average of experimental triplicates performed on the RNA samples from (B)]. (B) Northern blot for mature let-7g. (C) Confirmation of Lin28 knockdown using Lin28-SI2 on samples analyzed in (D). (D) Changes in mature miRNA levels upon knockdown of Lin28 as analyzed by quantitative PCR. (E) Levels of pri-let-7g upon knockdown of Lin28 in P19 cells. (F) Levels of the pluripotency markers Oct-4 and Nanog in P19 cells transfected with either control siRNA or Lin28-si. In (C) to (F), error bars represent SEM with N = 3.

A Lin28 homolog, Lin28B, is overexpressed in human hepatocellular carcinoma as well as in several cancer cell lines (13). Two isoforms of Lin28B, differing in their 5′ exons, have been reported. The short isoform (Lin28B-S) preserves the two retroviral-type CCHC zinc-finger motifs present in the long isoform (Lin28B-L) but contains a truncated cold-shock domain. Lin28B-L overexpression induces cancer cell proliferation, whereas Lin28B-S overexpression has no effect (13). We found that Lin28B-L potently inhibited the processing of pri-let-7g (fig. S8), whereas Lin28B-S did not. Hence, the previously reported oncogenic properties of Lin28B may be mediated, at least in part, through blockade of let-7 processing. It is possible that Lin28 and Lin28B may require both the cold-shock domain and CCHC zinc fingers for blocking activity. Lin28 and Lin28B are the only animal proteins known to contain both of these domains (15).

Our results show that Lin28 is necessary and sufficient for blockade of pri-miRNA processing of let-7 family members both in vitro and in vivo. There are several possible reasons why ES and EC cells possess a mechanism for posttranscriptional regulation of let-7 miRNA expression. First, posttranscriptional activation of miRNA processing would allow for rapid induction of several let-7 miRNAs by down-regulation of a single factor. Second, disruption of DGCR8, a dsRNA binding protein and essential component of the Microprocessor complex, interferes with ES cell differentiation, which suggests that activation of miRNAs may be important for silencing the self-renewal machinery (16). It has been suggested that posttranscriptional control could prevent even small amounts of let-7 from being produced in ES and EC cells, tightly maintaining the undifferentiated state (7). Third, posttranscriptional control of miRNA expression could serve as a means for dissociating expression patterns of intronic miRNAs from expression patterns of their host transcripts.

The precise mechanism by which Lin28 blocks miRNA processing as well as the range and determinants of its substrate selectivity are unknown. Our data suggest that Lin28 has a preference for selectively blocking the processing of let-7 family pri-miRNAs at the Microprocessor step. However, we cannot rule out the possibility that Lin28, alone or in concert with other factors, may block other pri-miRNAs in different physiological contexts. Others have reported that miRNA processing can also be regulated at the Dicer step, when pre-miRNAs are cleaved to their mature form (5, 8). Additional factors may yet be discovered that posttranscriptionally regulate miRNA processing. Lin28 is predominantly localized to the cytoplasm, although it can also be found in the nucleus (10, 11); Lin28B is translocated into the nucleus in a cell cycle–dependent fashion (13). Lin28 may posttranscriptionally regulate miRNA processing in embryonic cells in a cell cycle–specific manner.

Recently, Lin28 was used in conjunction with Nanog, Oct-4, and Sox2 to reprogram human fibroblasts to pluripotency (14). Our data thus suggest that modulating miRNA processing may contribute to the reprogramming of somatic cells to an embryonic state. Additionally, global inhibition of miRNA processing by knockdown of the Drosha component of the Microprocessor was shown to promote cellular transformation and tumorigenesis; this phenotype was found to be, in large part, due to loss of let-7 expression (17). Let-7 has been reported to play a tumor suppressor role in lung and breast cancer by repression of oncogenes such as Hmga2 (18) and Ras (19, 20). We suggest that disruption of let-7 processing by activation of Lin28 could promote the oncogenic phenotype. Notably, several human primary tumors show a general lack of correlation between expression of pri-miRNAs and the corresponding mature species (7, 21). This suggests that a block in miRNA processing may contribute to the low miRNA expression observed in many human cancers (22). Future study of Lin28 promises to reveal how miRNA processing contributes to the dedifferentiation that accompanies both somatic cell reprogramming and oncogenesis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1154040/DC1

Materials and Methods

Figs. S1 to S8

Table S1

References

References and Notes

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