Research Article

A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA

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Science  03 Aug 2001:
Vol. 293, Issue 5531, pp. 834-838
DOI: 10.1126/science.1062961

Abstract

The 21-nucleotide small temporal RNA (stRNA) let-7regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Two small temporal RNAs (stRNAs),lin-4 and let-7, regulate the timing of development in Caenorhabditis elegans(1–3). stRNAs encode no protein, but instead appear to block the productive translation of mRNA by binding sequences in the 3′-untranslated region of their target mRNAs (1, 2, 4–11).let-7 is present in most if not all bilaterally symmetric animals, including Drosophila melanogaster and humans (12). In Drosophila, let-7 first appears at the end of the third larval instar, accumulates to high levels in pupae, and persists in adult flies (12).

The mechanism by which stRNAs are synthesized is unknown. The ∼21-nucleotide (nt) let-7 RNA has been proposed to be cleaved from a larger precursor transcript (12). The generation of small RNAs from a longer, structured precursor—double-stranded RNA (dsRNA)—is an essential feature of the RNA interference (RNAi) pathway, raising the possibility that stRNAs are generated by mechanisms similar to the initial steps in RNAi and suggesting that enzymes such as the Drosophila protein Dicer might play a role in generating stRNAs (13–18).

A candidate RNA for the Drosophila let-7 precursor.

Examination of the developmental expression of let-7 inDrosophila revealed a candidate for a let-7precursor RNA, let-7L (19). let-7L was detected at the end of the third larval instar and at the beginning of pupation, the same developmental stages where let-7 itself is first expressed (Fig. 1A) (12). Consistent with the transcript being alet-7 precursor, the amount of let-7L RNA declined as let-7 accumulated. let-7L RNA was slightly shorter than a 76-nt RNA standard. Previous analysis of the genomic sequence flanking Drosophila let-7 led to the proposal that a 72-nt RNA hairpin might be a let-7 precursor (12) (Fig. 1B).

Figure 1

Pre-let-7 in vivo and in vitro. (A) Northern hybridization analysis of let-7expression during the Drosophila life cycle. The hybridization probe was complementary to both let-7 and the putative let-7 precursor RNA indicated in (B). Ethidium bromide staining of 5S ribosomal RNA demonstrates that approximately equal amounts of total RNA were loaded in each lane.let-7 detected in C. elegans total RNA has an apparent mobility 1 nt longer than that detected in flies. (B) Schematic of the proposed Drosophilapre-let-7 RNA (12), indicating the regions of complementarity to the primer extension probes used in this study. The sequence of mature let-7 within the precursor is indicated in red. (C) Northern hybridization analysis oflet-7 RNA in Drosophila pupae, embryos, and cultured S2 cells, as well as in cultured human HeLa cells. Two dashes indicate the positions of the let-7 doublet. The amount of RNA loaded in each lane is indicated at the top. (D) Primer extension with probe 1 to analyze total RNA fromC. elegans, Drosophila early and unstaged pupae, and cultured human HeLa cells grown in suspension. Primer extension of a synthetic RNA with the sequence indicated in (B) (left lane) and primer extension in the presence of 2′,3′-terminal dideoxynucleotides (indicated at right) provide markers. Solid arrowheads indicate primer extension products diagnostic of pre-let-7, and open arrowheads indicate primer extension products diagnostic of Drosophila and humanlet-7. An arrow indicates the primer extension product diagnostic of C. elegans let-7. Two primer extension products were detected for Drosophila and humanlet-7, consistent with previous S1 nuclease mapping (12) that suggests that both Drosophila and human let-7 exist in 21- and 22-nt forms that differ at their 5′ ends. Alternatively, the longer extension product may represent addition of one untemplated nucleotide by reverse transcriptase. (E) Primer extension analysis of an in vitro pre-let-7 processing reaction.Drosophila embryo lysate was incubated in a standard cell-free RNAi reaction with or without (Ø) pre-let-7 for the time indicated. The RNA was then deproteinized and analyzed by primer extension with probe 1. The identity of the 5′ ends of the extension products (indicated at right) was determined by reverse transcription in the presence of dideoxynucleotides.

let-7 is also expressed in human tissues (12) and in cultured human HeLa cells, but not in Drosophila embryos or cultured Drosophila S2 cells (Fig. 1C). Primer extension analyses confirmed that the mature let-7 RNA detected by Northern hybridization was bona fide let-7 (20). Primer extension products corresponding to the 5′ ends of maturelet-7 RNAs were detected in total RNA from early and unstaged Drosophila pupae and from human HeLa cells (Fig. 1D). Primer extension analysis of total RNA from unstaged worms (Fig. 1D), as well as Northern hybridization experiments (Fig. 1A), indicated that worm let-7 is 1 nt longer than that in flies and humans. In early pupae, primer extension analysis also detected three longer extension products. The major, middle product and the less abundant, lower product comigrate with primer extension products templated by a synthetic 72-nt RNA corresponding to putative pre-let-7 (Fig. 1, B and D). This longer transcript from early pupae (Fig. 1A) has the same 5′ end as the 72-nt let-7precursor proposed by Ruvkun and co-workers (Fig. 1B) and is therefore a good candidate for a let-7 precursor RNA (12).

let-7 maturation in vitro.

To determine if the let-7L RNA detected in vivo was, in fact, the direct precursor of mature let-7, we tested processing of the proposed pre-let-7 stem-loop RNA intolet-7 in Drosophila embryo lysates, which contain no detectable let-7 RNA (Fig. 1C) (12). These lysates recapitulate RNAi in vitro (21), allowing us to ask if the proposed precursor RNA was cleaved into maturelet-7 by an RNAi-like mechanism. The 72-nt RNA (Fig. 1B) was incubated with Drosophila embryo lysate for various times, then assayed for the production of let-7 by primer extension (Fig. 1E). As seen in vivo, mature let-7 RNA accumulated in the cell-free reaction. Thus, an RNA corresponding to the proposedlet-7 precursor was converted to an RNA with precisely the same 5′ ends as authentic let-7 by one or more factors in the Drosophila embryo lysate.

Only let-7 RNA, not its complement, has been detected in vivo in worms, flies, and human tissues (22, 12). Thus, we expected that bona fide let-7 maturation in vitro would be asymmetric, yielding only let-7 and not small RNAs complementary to let-7, such as antisense let-7. In contrast, processing of long, dsRNA by the RNAi pathway is symmetric, yielding double-stranded 21- to 22-nt RNAs. Therefore, we asked if processing of the proposed pre-let-7 RNA in vitro was symmetric or asymmetric, yielding let-7 but not its complement. We prepared four pre-let-7 RNAs by in vitro transcription, each uniformly labeled with a different α-32P–nucleotide—adenosine 5′-triphosphate (ATP), cytidine 5′-triphosphate, guanosine 5′-triphosphate, or uridine 5′-triphosphate—and incubated them separately in an in vitro reaction (23). Since let-7 contains no cytosine, accurate in vitro processing of pre-let-7 should produce a 21- to 22-nt product for RNAs labeled at A, G, or U but not at C. A product of the appropriate size for let-7 was produced for pre-let-7 transcripts labeled at A, G, and U (Fig. 2A). No 32P-labeled product accumulated from the 32P-C-labeled pre-let-7RNA; the faint ∼22-nt band visible in the 32P-C lanes does not correspond to a processing product because it was present before the reaction was initiated (0 min lane) and disappeared upon incubation in the Drosophila embryo lysate. Although pre-let-7 RNA continued to disappear with incubation in the lysate, mature-let-7 production rapidly reached a plateau. Because single-stranded 21-nt RNAs are generally unstable in the embryo lysate (24, 25), this likely reflects degradation of let-7 in the lysate, which may lack factors required for let-7 stabilization and function. Nonetheless, it is remarkable that let-7 RNA accumulates at all, because exogenous, single-stranded, 21-nt RNAs are degraded by the lysate within minutes.

Figure 2

In vitro processing of Drosophilapre-let-7. (A) Time course of in vitro processing for in vitro–transcribed pre-let-7 RNAs labeled with the α-32P nucleotide indicated at the bottom of the figure. Molecular size markers are from a complete T1 digestion of a uniformly labeled RNA. Because the electrophoretic mobility for small RNAs is a function of sequence and terminal structure as well as of length, both synthetic 21-nt let-7 RNA (not shown) and in vitro–processed let-7 migrate at ∼23 nt relative to these markers. (B) Northern hybridization analysis with probes 2, 3, and 4 (see Fig. 1B) of the products of an in vitro pre-let-7 processing reaction. The arrow indicates the position of mature let-7. The same filter was stripped and reprobed for the experiments in each panel. (C) Quantitation of the data in (B). The amount of hybridization of the probe to the 21- to 22-nt RNA products was quantified and was normalized to the amount of hybridization of the probe to the unreacted precursor remaining at 3 hours to correct for differences in hybridization efficiencies among the three probes.

Next, we analyzed the products of an in vitro reaction by Northern hybridization (Fig. 2B) using three different deoxyoligonucleotide probes (Fig. 1B). Probe 2 was entirely complementary to maturelet-7. Probe 3 was complementary to the first 21 nt of the precursor and therefore only partially complementary to maturelet-7. Control experiments showed that probe 3 detected mature let-7 substantially less well than probe 2, whereas probe 3 detected as well or better than probe 2 products derived from the precursor sequence that is 5′ to the region encodinglet-7 (23, 26). Finally, probe 4 was complementary to the side of the stem of the precursor opposite the portion encoding let-7 (Fig. 1B). Thus, probe 4 should detect the products of symmetric processing of the precursor RNA. Control experiments demonstrated that probe 4 readily detected synthetic antisense let-7 RNA, but not let-7itself (23, 26). Northern hybridization experiments were quantified by determining the amount of each probe that hybridized to the region of the blot corresponding to the ∼21-nt reaction product and, as a control for hybridization efficiency, the amount of hybridization of each probe to the unreacted precursor remaining at 3 hours, because the full-length precursor is perfectly complementary to all three probes (Fig. 2C). Probe 2, which is complementary tolet-7, readily detected an RNA that accumulated with time. In contrast, probe 3 detected only weakly an RNA that accumulated over the course of the reaction, consistent with it detecting by partial hybridization mature let-7 but not reaction products derived from the region of the precursor 5′ to the let-7 sequence. Most important, probe 4, which was designed to detect reaction products like antisense let-7, did not detect products that accumulated upon incubation of pre-let-7 in the lysate (Fig. 2, B and C). These data strongly imply that symmetric processing products such as antisense let-7 are either not generated at all or are far less stable than let-7 in the in vitro reaction. Thus, the in vitro reaction displays the same specificity and asymmetry that characterize let-7 biogenesis in vivo.

It remained possible that the mechanisms of cleavage in vitro and in vivo differ. To assess the type of ribonuclease (RNase) that might be responsible for pre-let-7 processing, both in vitro and in vivo, we analyzed the 5′ and 3′ ends of both the let-7generated by the in vitro processing reaction and the let-7from pupae (Fig. 3) (27). Treatment with periodate, followed by β-elimination, of either RNA from the in vitro processing reaction or total pupal RNA increased the apparent mobility of let-7 by nearly 2 nt, a change diagnostic of RNAs bearing 2′,3′-terminal hydroxyl groups (Fig. 3, A and C). Treatment with calf intestinal phosphatase (CIP) of in vitro–generated let-7 or pupal RNA decreased the apparent mobility of let-7 by 1 nt, consistent with the removal of a charged phosphate group (Fig. 3, B and C). Furthermore, treatment of the CIP-treated RNA with polynucleotide kinase and ATP restored its original mobility, demonstrating thatlet-7 contains a monophosphate (28). Becauselet-7 contains 2′- and 3′-terminal hydroxyls, this single phosphate must be at its 5′ end. Thus, let-7 produced by in vitro processing and let-7 isolated from pupae have the same terminal structure: a 5′ monophosphate and 2′- and 3′-terminal hydroxyls. Notably, such termini are characteristic of the products of cleavage of dsRNA by RNase III (29).

Figure 3

Analysis of the ends of let-7produced in vitro and in vivo. let-7 RNA was detected by Northern hybridization. (A) Drosophilapre-let-7 RNA was processed in vitro for the times indicated and analyzed with and without periodate treatment followed by β-elimination. The apparent electrophoretic mobility of untreatedlet-7 (arrow) is increased by ∼2 nt (arrowhead) upon periodate treatment. (B) Drosophilapre-let-7 was processed in vitro for the times indicated and analyzed with and without calf intestinal phosphatase (CIP) treatment. The apparent mobility of let-7 (arrow) is reduced by ∼1 nt (arrowhead) upon CIP treatment. (C) Analysis of maturelet-7 in total pupal RNA (Ø) or in total pupal RNA treated with CIP (CIP) or periodate followed by β-elimination (β). Control lanes correspond to a synthetic 21-nt let-7 RNA bearing a 5′ monophosphate and 2′- and 3′-terminal hydroxyl groups (Ø) or the same RNA treated with CIP (CIP) or periodate followed by β-elimination (β). The apparent mobility of pupal let-7(arrows) is reduced by ∼1 nt (open arrowheads) when treated with CIP and increased by ∼2 nt (filled arrowheads) when treated with periodate.

The small interfering RNAs (siRNAs) that mediate RNAi also bear a 5′ monophosphate and 2′- and 3′-terminal hydroxyls (25). InDrosophila, siRNA duplexes are produced by the cleavage of long dsRNA by the enzyme Dicer (18). Cleavage by Dicer is thought to be catalyzed by its tandem RNase III domains. Only two types of RNase III enzymes are predicted to occur in Drosophila: Drosha (30) and Dicer. Dicer is the only RNase III domain protein in the publicly available sequence of the Drosophilagenome that contains an ATP-binding motif, the DEAD-box RNA helicase domain (18). Cleavage of dsRNA by Dicer is strictly ATP-dependent (18). Figure 4A shows that cleavage of pre-let-7 into maturelet-7 in Drosophila embryo lysates also required ATP. Taken together, the chemical structure of mature let-7RNA in vitro and in vivo and the ATP dependence of pre-let-7processing in vitro strongly implicate Dicer in let-7maturation. However, we note that expression of Dicer protein inDrosophila larvae or pupae has not yet been demonstrated, although the RNAi pathway, which requires Dicer, functions in larvae and pupae (31).

Figure 4

Evidence for a role for Dicer protein in pre-let-7 processing in vitro and in vivo. (A) In vitro processing of pre-let-7 requires ATP. The predictedDrosophila pre-let-7 RNA (50 nM) was incubated with Drosophila embryo lysate in vitro with (mock-depleted) and without (ATP-depleted) ATP for the times indicated. Production oflet-7 was monitored by Northern hybridization. (B) Semiquantitative RT-PCR assays for Dicer and actin mRNA levels in HeLa cells transfected with buffer (untreated), control siRNA, or human Dicer siRNA. The PCR primers span one (Dicer) or two (actin) introns. Therefore, the PCR products correspond to amplification of mRNA, not genomic, sequences. (C) siRNA-targeted degradation of human Dicer mRNA leads to the accumulation of let-7L in cultured human HeLa cells. Total RNA from untreated (no siRNA), control siRNA-treated, or human Dicer siRNA-treated HeLa cells, and from HeLa cells grown in suspension and from early Drosophila pupae was analyzed by primer extension to detect let-7L and mature let-7. Primer extension products diagnostic of let-7, the predicted human and Drosophila let-7 precursors (arrowheads), and an extended stem form of the precursor (*) are indicated. (D) Proposed structure of the shorter and extended human let-7LRNAs detected in (C). The sequence of mature let-7 is shown in red. The 5′ ends of the primer extension products corresponding to the shorter precursor (arrowhead) and the extended precursor (arrowhead with asterisk) are indicated at right. The structure was predicted with the RNA-folding algorithm mFold 3.1 (42, 43). If the unpaired nucleotides at positions 7, 8, 16, and 17 are allowed to form G • U base pairs, then the stem may comprise as many as 32 consecutive base pairs interrupted only by the unpaired uracil residues at positions 12 (the 5′ end of mature let-7) and 87.

A more stringent test for a role for Dicer in pre-let-7 processing would be to assay let-7production in flies lacking Dicer protein. However, mutant alleles of Dicer have yet to be identified in Drosophila. As an alternative approach, we used a recently reported sequence-specific method in which cultured mammalian cells are transfected with synthetic 21-nt siRNA duplexes to suppress gene expression (32). Because they are <30 base pairs long, the siRNA duplexes do not trigger the sequence-nonspecific responses that complicate standard dsRNA-induced interference in mammalian cells.

The RNAi enzyme Dicer is required for maturation of humanlet-7 RNA.

We used this method to evaluate the role of the human ortholog of Dicer (Helicase-MOI) in let-7 biogenesis. Human Dicer was identified by its unique domain structure, comprising an NH2-terminal DEXH-box ATP-dependent RNA helicase domain, PAZ domain, tandem RNase III motifs, and COOH-terminal dsRNA-binding domain, and by its sequence homology toDrosophila Dicer (18, 33,34). HeLa cells were transfected with a single, synthetic siRNA duplex containing 19 nt of the coding sequence of human Dicer mRNA, beginning at position 183 relative to the start of translation (35). Three days after transfection, total RNA was prepared from the cells and analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) for Dicer and actin mRNA levels and by primer extension for the presence of let-7 (Fig. 4, B and C). The level of Dicer mRNA in the Dicer siRNA-treated cells was four- to sixfold lower than in the control samples, whereas actin mRNA levels were unchanged (Fig. 4B). Separate controls showed that ∼70 to 80% of the cells were transfected. Thus, the observed decrease in Dicer mRNA levels demonstrates that the Dicer siRNA induced substantial degradation of Dicer mRNA in the fraction of the cells that were successfully transfected.

Transfection of HeLa cells with the siRNA duplex corresponding to human Dicer, but not the control siRNA duplex, led to the accumulation of a longer let-7–containing RNA, let-7L: Primer extension analysis of RNA from cells transfected with the Dicer siRNA detected an RNA with a 5′ end ∼7 nt and ∼11 to 12 nt upstream of the mature let-7 product (Fig. 4C). These products are consistent with the accumulation of the predicted humanlet-7 precursor RNA (12) and with a longer form of this precursor containing an extended stem (Fig. 4D). The mature human let-7 RNA was readily detected in control cells, but not in the cells transfected with the Dicer siRNA duplex (Fig. 4C), providing additional evidence for a role for Dicer in let-7maturation. These findings, together with our in vitro data, provide strong evidence that Dicer protein function is required for the maturation of let-7. Thus, the RNAi and stRNA pathways intersect; both require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression (Fig. 5). The two pathways must also diverge after the action of Dicer, because siRNA duplexes generated from long, dsRNA direct mRNA cleavage, whereas the single-stranded stRNA let-7 represses mRNA translation.

Figure 5

The RNAi and stRNA pathways intersect. In this model, transcription of pre-let-7 and other stRNA precursors is regulated developmentally. Dicer and perhaps other proteins act on these pre-stRNAs to yield mature, single-stranded stRNAs that repress mRNA translation. In RNAi, Dicer cleaves long, dsRNA to yield siRNA duplexes that mediate targeted mRNA destruction.

Recently, Mello and co-workers have shown that the Dicer homolog Dcr-1 is required for both lin-4 and let-7function in C. elegans (36). Thus, Dicer is likely to have a broad role in the biogenesis of stRNAs and perhaps other small regulatory RNAs. Furthermore, mutations in theArabidopsis homolog of Dicer, SIN-1/CARPEL FACTORY(SIN1/CAF), have dramatic developmental consequences (37–39). Perhaps SIN1/CAF protein in plants, like Dicer in bilateral animals, processes structured RNA precursors into small RNAs that regulate development.

Pre-let-7 is processed asymmetrically to yield onlylet-7. We do not yet know what structural or sequence features of pre-let-7 determine its asymmetric cleavage. RNase III enzymes cleave perfectly paired dsRNA on both strands, producing a pair of cuts, one on each strand, displaced by two nucleotides. For the R1.1 RNA hairpin of T7 bacteriophage, internal loops and bulges constrain the Escherichia coli RNase III dimer to cut only one strand of the stem (40). The proposedlet-7 precursor contains such an internal loop at the site of 5′ cleavage. It is possible that if the stem were uninterrupted by such distortions, a pair of 21- to 22-nt RNAs might be generated, rather than the single stRNA let-7. If so, it might be possible to design stem-loop RNA precursors that produce an siRNA duplex. The hope is that such an siRNA duplex, generated in vivo in a specific cell type or at a specific developmental stage, would be able to target an mRNA for destruction by the RNAi machinery, thereby extending the utility of RNAi to the study of mammalian development.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: phillip.zamore{at}umassmed.edu

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