ecd1 is a temperature-sensitive mutation in the ecdysoneless gene (Garen, 1977) that impairs the biosynthesis of ecdysone at restrictive temperature (29°C). To test whether reduced ecdysone at the end of the L3 stage affects the expression of let-7 RNA, ecd1 animals were transferred to 29°C at various times during the L3 stage to interfere with the generation of this ecdysone pulse. At the permissive temperature (20°C), ecd1 animals pupariate around 230 h after egg laying. This time of PF was used as a reference for defining the duration of the L3 stage of ecd1 animals. ecd1 animals were synchronized at egg laying and reared at 20°C until they were transferred to 29°C in the early and mid L3 stage. These developmental stages are approximate, because ecd1 animals grow more slowly and less synchronously than the wild type. The majority of ecd1 animals that were upshifted in the early L3 stage remained as larvae (98%), and these ecd1 retarded larvae were harvested at various times relative to PF of ecd1 animals maintained at 20°C. ecd1 animals upshifted in the mid-L3 stage produced a mixture of pupariating (40%) and nonpupariating (60%) individuals, and these were harvested separately. Wild-type animals reared at 20°C were upshifted as late L3 larvae for comparison. Using Northern analysis of total RNA, it was found that let-7 RNA is marginally expressed in ecd1 animals; these animals fail to pupariate due to the absence of ecdysone. In contrast, let-7 is expressed at much higher levels in pupariating ecd1 animals. This correlation between PF and let-7 expression in ecd1 animals suggests that these two events are triggered by the same pulse of ecdysone. This could reflect that the induction of let-7 is mediated by the ecdysone signaling pathway. Alternatively, let-7 expression could be activated by another developmental signal associated with PF and/or progression through metamorphosis (Sempere, 2002).
In ecd1 pupae maintained at 29°C for more than 6 h after PF, levels of let-7 RNA were reduced compared with the wild type, suggesting that a prolonged pulse of high ecdysone titer throughout pupal development may be required to sustain let-7 expression. A similar reduction of let-7 RNA levels was also observed in ecd1 pupae transferred at different times after PF (Sempere, 2002).
It is inferred from the absence of let-7 RNA in nonpupariating ecd1 mutants that ecdysone is required for let-7 expression. To test this supposition, organ cultures from third instar larvae were incubated with ecdysone. Late L3 larvae were dissected to expose the internal organs to the medium. After washes to remove ecdysone circulating in the hemolymph, organs were cultured in Schneider's medium with or without 5 microM ecdysone, for various lengths of time (0-20 h). Levels of let-7 RNA were examined by Northern analysis of total RNA recovered from these harvested organs. let-7 is already expressed at relatively low levels at the time of dissection (0 h), presumably due to the rising titer of ecdysone. let-7 expression remains at this low level in organs incubated without ecdysone during the first 12 h, and levels of let-7S and its precursor form (let-7L) decrease substantially at later times. In contrast, levels of let-7 RNAs increase after 12 h in organs incubated with ecdysone. This increased accumulation likely results from increased transcription since levels of let-7S and let-7L increase coordinately (Sempere, 2002).
The early gene, Broad-Complex (BR-C), is located at the top of the regulatory hierarchy in the ecdysone pathway and plays an essential role in regulating the expression of downstream targets. BR-C encodes four isoforms of a zinc finger transcription factor (Z1-Z4) that not only control directly the expression of late genes, but that are also required for full expression of other early genes. To test whether BR-C is involved in mediating the response of let-7 expression to ecdysone pulses, the levels of let-7 RNA were determined in animals homozygous for a BR-C null mutation. Homozygous npr6 animals are deficient in all four BR-C isoforms (Z1-Z4), rendering them unresponsive to the ecdysone pulse at the end of the L3, and hence they fail to pupariate. npr6 animals remain as larvae for about 5-10 days after the normal time of pupariation and then die. npr6/npr6 and npr6/+ animals were synchronized and harvested at various times relative to the time of PF (of the npr6/+ animals). let-7 RNA was detected at very low levels in npr6/npr6 animals, compared with npr6/+ siblings, indicating that BR-C is required in vivo to activate let-7 expression in response to ecdysone. Interestingly, there is a slight increase of let-7 expression in npr6/npr6 animals 48 h after 'PF', possibly corresponding to a rise of ecdysone titer. This suggests that other components of the ecdysone pathway could play a secondary role in let-7 expression, independent of BR-C (Sempere, 2002).
The above in vivo experiments suggest that ecdysone signaling is required for let-7 expression, and that there is at least one intermediate player between ecdysone and let-7 expression, the BR-C transcription factor. Next it was asked whether ecdysone directly activates let-7, that is, does ecdysone act on a tissue and trigger the expression of let-7 in that tissue, instead of initiating a signaling cascade resulting in the expression of let-7 in another tissue? To address this, the ability of ecdysone to initiate let-7 expression was examined in cultured S2 cells, which are known to be responsive to ecdysone. S2 cells were incubated in the presence of ecdysone at a final concentration of 5 microM for 6-62 h, and harvested every 6 h. let-7 RNA was first detected at approximately 18 h after incubation with ecdysone, increased in level from 24 to 42 h, and reaching a plateau at 42-54 h, and decreasing thereafter. let-7 RNA was not detected in untreated S2 cells. This result indicates that ecdysone induces let-7 expression directly in cells to which it is applied. The long delay between the ecdysone primary action and let-7 activation in S2 cells is consistent with the participation of intermediate regulators (Sempere, 2002).
To test whether ecdysone is required to sustain let-7 expression in S2 cells, a pulse-chase experiment was carried out. S2 cells were incubated for 24 h in the presence of 5 microM ecdysone, and then divided into two cultures. From one culture, the medium was removed and replaced by an ecdysone-free medium. The other culture was kept in 5 microM ecdysone for the remainder of the experiment. Northern blot analysis of total RNA showed a decrease in let-7 RNA levels in S2 cells after removal of ecdysone from the medium, compared with the cells maintained with ecdysone. The levels of let-7S and let-7L coordinately decreased. These results indicate that ecdysone is required for both the initiation and maintenance of let-7 transcription in S2 cells (Sempere, 2002).
BR-C is involved in relaying the ecdysone signal to trigger let-7 expression in vivo. To determine whether a similar BR-C-dependent pathway mediates let-7 activation in S2 cells, BR-C activity was inhibited by RNA interference (RNAi). S2 cells were transfected with a dsRNA corresponding to a conserved sequence in all four BR-C transcript isoforms. Control cells were transfected with a nonspecific dsRNA (from an unrelated C. elegans sequence). After 30 min of incubation with dsRNA, cells were treated with 5 microM ecdysone. In parallel, non-transfected cells were also treated with 5 microM ecdysone. S2 cells were harvested for 32, 40, and 48 h after the addition of ecdysone, and total RNA was analyzed by Northern blotting. The profile of let-7 expression was very similar in nontransfected cells and in cells transfected with nonspecific dsRNA. However, let-7 expression was dramatically reduced in cells transfected with dsRNA against BR-C. This indicates that BR-C is required for ecdysone-dependent let-7 expression in S2 cells. Since levels of let-7S and let-7L RNA coordinately decrease when BR-C activity is reduced by RNAi, BR-C likely affects transcription of let-7 in response to ecdysone. The residual let-7 expression in BR-C RNAi cells could be due to the ineffecient uptake of the dsRNA or the incomplete inhibition of BR-C activity. Alternatively, other components of the ecdysone pathway could contribute to let-7 expression, consistent with the residual let-7 expression observed in npr6 mutant animals (Sempere, 2002).
The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of C. elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development. This study asks whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an ~500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. It is concluded that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway (Bashirullah, 2003).
Mapping of the miR-125 sequence to the Drosophila genome reveals that it is encoded adjacent to let-7, with the precursor sequences located ~300 bp from one another at position 36F in the polytene chromosomes. This close physical proximity combined with the precise temporal coordination of let-7 and miR-125 expression raises the interesting possibility that they might be expressed from a common precursor RNA. To test this possibility, a fragment encompassing this region was used as a probe for Northern blot hybridization using RNA samples from staged Drosophila as well as RNA samples from Kc cells treated with 20E. An ~500-nt RNA can be detected in synchrony with the appearance of let-7 and miR-125 RNA in late third instar larvae, early pupae, and Kc cells treated with 20E for 25 h. This expression, however, is transient, as the ~500-nt RNA is not detected at later stages during Drosophila development. The size and temporal expression pattern of this RNA is consistent with the proposal that it acts as an initial common precursor for the synthesis of both let-7 and miR-125. Moreover, the transient expression of this precursor indicates that the let-7/miR-125 gene cluster is only expressed during early Drosophila metamorphosis, while the more stable 21- to 22-nt products persist through adult stages (Bashirullah, 2003).
Thus, let-7 and miR-125 are induced in late third instar larvae and prepupae in a temporal pattern that mirrors that of a known ecdysone primary-response mRNA, E74A. In spite of this tight temporal correlation, however, the data argue against a role for ecdysone signaling in controlling the timing of let-7 and miR-125 induction in Drosophila. Little effect on their induction is seen when EcR function is blocked by RNAi, and no induction is seen of let-7 or miR-125 by the physiologically active form of the hormone, 20E, in cultured larval organs under conditions where E74A is abundantly expressed. let-7 and miR-125 induction by 20E in Kc cells is delayed by at least 10 h relative to that of the primary-response E74A mRNA. This delay suggests that these microRNAs are expressed as either a secondary-response to the hormone or as an indirect consequence of the 20E-induced differentiation program in these cells. Regardless, this pattern of expression is distinct from the precise coordinate expression of E74A, let-7, and miR-125 seen in vivo, indicating that a different mechanism is responsible for miRNA induction in the animal (Bashirullah, 2003).
One study [Sempere (2002)] of let-7 regulation in Drosophila arrives at a different conclusion from that reached in this study. Much of their data, however, is consistent with the results presented in this study. Sempere presents a time course of let-7 induction by 20E in S2 tissue culture cells that is very similar to that reported in this study in Kc cells, although Sempere does not use a primary-response gene as a temporal marker for direct induction by the ecdysone-receptor complex. As they point out, their results with the ecd1 ecdysone-deficient mutant and npr6 mutant are difficult to interpret because the lack of let-7 expression in these mutants could simply be attributed to their developmental arrest as third instar larvae and inability to initiate puparium formation. The most significant contradiction with their data is their results with cultured larval organs; these show increased levels of let-7 RNA in the presence of 20E. The Sempere experiment is, however, difficult to interpret. The 0-h time point has let-7 RNA present, as do the 8- and 12-h time points in the absence of 20E, indicating that the organs used for this study were taken at a time when let-7 was already expressed. It is possible that 20E could be stabilizing or maintaining let-7 expression in these organs. The authors provide no marker for a known primary-response to ecdysone, so the timing of their induction cannot be interpreted. The authors also take their earliest collection (8 h) at the time when primary-response genes are starting to be repressed, several hours after initial gene induction (Bashirullah, 2003).
Some miRNAs map within close proximity to one another, forming apparent gene clusters in C. elegans, Drosophila, and humans. A similar arrangement is found for let-7 and miR-125 in Drosophila, that map within ~300 bp of one another on the left arm of the second chromosome. In addition, a precursor RNA is detected that could encode both miRNAs, providing a means of explaining their tight temporal coordination. This observation is consistent with the recent identification in HeLa cells of relatively long primary transcripts (pri-miRNAs) that contain multiple ~70-nt stem-loop microRNA precursors, and indicates that similar precursors can be expressed in a developing organism. Although expression of some miRNAs may be regulated by multiple processing steps, the low abundance and transient appearance of the let-7/miR-125 relatively long primary transcripts (pri-miRNA) and shorter pre-miRNA forms indicate that transcription of the primary transcript could be largely responsible for the precise temporal appearance of the mature miRNAs (Bashirullah, 2003).
Interestingly, miR-125 provides the best match in the Drosophila genome to a second small heterochronic RNA in C. elegans, lin-4. The two mismatches between lin-4 and miR-125, however, are sufficient to render miR-125 undetectable on Northern blots of Drosophila RNA using lin-4 sequences as a probe. Moreover, it is likely that these sequence differences have an impact on the specificity of miR-125 interactions with possible target sequences in 3'UTRs. The functional significance of this sequence similarity thus remains to be determined (Bashirullah, 2003).
These studies leave unanswered the question of what induces let-7 and miR-125 expression at the onset of Drosophila metamorphosis. The timing of the upregulation of these miRNAs in staged animals argues that it is responding to a temporal signal that occurs in parallel with the well-characterized 20E/EcR/USP signaling pathway. Because of this tight correlation, and the widespread expression of let-7 RNA in Drosophila (Sempere, 2002), the most likely candidate regulator would be a hormone that acts through a receptor other than EcR. This conclusion is consistent with several recent studies that have provided indirect evidence of other hormone signaling pathways in Drosophila (Bashirullah, 2003).
Many genes are coordinately upregulated in mid-third instar larvae, when the Adh gene switches from its larval to adult promoter, and the larval salivary gland switches its genetic program from the ng/Pig genes to the Sgs glue genes. The temporal signal for this mid-third instar transition remains undefined, but could comprise a 20E/EcR-independent pathway. Similarly, the E74A and E75A early mRNAs are coordinately induced at some times during development when the ecdysteroid titer is thought to be low, arguing that another temporal signal may be responsible for this response. Finally, alpha-ecdysone, the precursor to 20E, is sufficient to drive furrow progression in Manduca eye primordia as well as stimulate optic lobe neuroblast proliferation, suggesting that this hormone, which is a poor activator of the EcR/USP complex, can act as temporal signal in this lepidopteran insect. Further studies should help to elucidate the roles of other ecdysteroids during insect development and provide a foundation for better understanding the temporal regulation of let-7 and miR-125 in Drosophila (Bashirullah, 2003).
It is interesting to note that the DAF-12 orphan nuclear receptor is required for the proper timing of let-7 induction in C. elegans. Genetic evidence suggests that DAF-12 is regulated by a steroid hormone under the control of the daf-9 cytochrome P450 gene. Perhaps more interesting from the perspective of this study, the Drosophila genome encodes an ortholog of DAF-12: DHR96. It is possible that functional studies of DHR96 in Drosophila will shed light on the regulation of the let-7/miR-125 gene cluster in this insect model system (Bashirullah, 2003).
lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, the developmental expression profile of 24 Drosophila miRNAs were characterized; seven miRNAs are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homolog of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo (Sempere, 2003).
The 24-h lag between the addition of Ecd to cultured S2 cells and the expression of mir-100 and mir-125 suggest that the initial Ecd signal activates mir-100 and mir-125 expression indirectly via intermediate regulators. One such intermediate could be BR-C, which is required for mir-100 and mir-125 expression in animals. To test whether BR-C activity is required for the Ecd-induced expression of mir-100 and mir-125 in S2 cells, BR-C activity was inhibited by RNAi using a 700-nucleotide dsRNA corresponding to a common region of all BR-C isoforms. S2 cells were incubated for 30 min with BR-C dsRNA or mock dsRNA, corresponding to unrelated C. elegans sequence. Then, the transfected and nontransfected cultures were treated with Ecd and harvested 32, 40, and 48 h later. The levels of mir-100 and mir-125 RNAs were considerably lower in BR-C RNAi cells as compared with nontransfected or mock RNAi cells. This result further supports the conclusion that BR-C is required to mediate the activation of mir-100 and mir-125 by an Ecd signal in vivo. This result also argues against the possibility that mir-100, mir-125, and let-7 RNAs were detected at very low levels in nonpupariating ecd1 and npr6 mutants simply because these mutant animals were arrested at a stage before mir-100, mir-125, and let-7 are normally upregulated (Sempere, 2003).
It should be noted that BR-C RNAi does not result in complete loss of mir-100 and mir-125 expression, suggesting that RNAi treatment is not fully effective. Consistent with an incomplete knockdown of BR-C by RNAi, miR-34 levels are unaffected by BR-C RNAi in Ecd-treated cells. Based on results with npr6 mutant animals, one would have expected that mir-34 expression would be derepressed by BR-C RNAi in Ecd-treated cells. Since BR-C activity may not have been completely eliminated by RNAi, the requirement for BR-C activity in the repression of mir-34 could not be assesssed in S2 cells (Sempere, 2003).
Interestingly, the sequence of mir-125 is quite similar to the sequence of lin-4, suggesting that they may be homologs. The evolutionary conservation of microRNAs such as mir-125/lin-4, mir-100, and let-7 implies the conservation of multiple complementary target sequences for each of the miRNAs. In C. elegans, lin-4 translationally downregulates LIN-14 and LIN-28 protein expression by base-pairing to partially complementary sites in the 3' UTR of their mRNAs. Translational repression of lin-14 and lin-28 activities by lin-4 are required for the temporal transition from early to late developmental programs, eventually leading to the adult differentiation of hypodermis and vulva. Although lin-28 is a member of an evolutionary conserved gene family, no obvious Drosophila or human homologs of lin-14 have been found and the targets of mir-125 in flies and vertebrates are yet to be identified (Sempere, 2003).
The finding that the mir-100, -125, and let-7 gene cluster is coregulated in Drosophila suggests that these three genes may act in concert to control the translation of target genes involved in adult morphogenesis and differentiation. mir-100, -125, and let-7 are quite distinct in sequence, and so they probably base-pair to distinct cognate binding sites in their target mRNAs. These three miRNAs could have unique target mRNAs that they repress in parallel, and/or they could act together to repress particular targets that contain the appropriate combination of cognate sites (Sempere, 2003).
With the exception of C. elegans, the chromosomal clustering of mir-100, mir-125/lin-4, and let-7 appears to be widely conserved in animal phylogeny. In the mosquito Anopheles gambiae, mir-100, -125, and let-7 are clustered within 5000 bp and oriented in the same direction. In vertebrates, mir-100, -125, and let-7 are similarly clustered, although with somewhat greater spacing. Remarkably, mir-100 and let-7 are located together in two distinct chromosomal locations within 500 and 2000 bp in the Puffer fish Takifugu rubripes, and within 700 bp and 5000 bp in humans. The conservation of the clustered arrangement of mir-100, mir-125, and let-7 suggests that important aspects of their regulation may also be evolutionary conserved (Sempere, 2003).
The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. In vivo and in vitro evidence is presented 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 (see Drosophila 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 (Hutvagner, 2001).
Two small temporal RNAs (stRNAs), lin-4 and let-7, regulate the timing of development in Caenorhabditis elegans. 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. let-7 is present in most if not all bilaterally symmetric animals, including Drosophila melanogaster and humans. 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 (Hutvagner, 2001).
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. 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 (Hutvagner, 2001),
Examination of the developmental expression of let-7 in Drosophila revealed a candidate for a let-7 precursor RNA, let-7L (Pasquinelli, 2000). 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. Consistent with the transcript being a let-7 precursor, the amount of let-7L RNA declines as let-7 accumulates. let-7L RNA is 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 (Pasquinelli, 2000; Hutvagner, 2001).
A let-7 homolog is also expressed in human tissues (Pasquinelli, 2000) and in cultured human HeLa cells, but not in Drosophila embryos or cultured Drosophila S2 cells. Primer extension analyses confirmed that the mature Drosophila let-7 RNA detected by Northern hybridization was bona fide let-7. Primer extension products corresponding to the 5' ends of mature let-7 RNAs were detected in total RNA from early and unstaged Drosophila pupae and from human HeLa cells. Primer extension analysis of total RNA from unstaged worms, as well as Northern hybridization experiments, 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. This longer transcript from early pupae has the same 5' end as the 72-nt let-7 precursor and is therefore a good candidate for a let-7 precursor RNA (Pasquinelli, 2000; Hutvagner, 2001).
To determine if the let-7L RNA detected in vivo is, in fact, the direct precursor of mature let-7, processing of the proposed pre-let-7 stem-loop RNA into let-7 was tested in Drosophila embryo lysates, which contain no detectable let-7 RNA (Pasquinelli, 2000). These lysates recapitulate RNAi in vitro, prompting the question of whether the proposed precursor RNA is cleaved into mature let-7 by an RNAi-like mechanism. The 72-nt RNA was incubated with Drosophila embryo lysate for various times, then assayed for the production of let-7 by primer extension. As seen in vivo, mature let-7 RNA accumulates in the cell-free reaction. Thus, an RNA corresponding to the proposed let-7 precursor is converted to an RNA with precisely the same 5' ends as authentic let-7 by one or more factors in the Drosophila embryo lysate (Hutvagner, 2001).
Only let-7 RNA, not its complement, has been detected in vivo in worms, flies, and human tissues (Pasquinelli, 2000). Thus, it is 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, it was asked if processing of the proposed pre-let-7 RNA in vitro is symmetric or asymmetric, yielding let-7 but not its complement. Four pre-let-7 RNAs were prepared by in vitro transcription, each uniformly labeled with a different alpha-32P-nucleotide (adenosine 5'-triphosphate (ATP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, or uridine 5'-triphosphate) and incubated separately in an in vitro reaction. 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. No 32P-labeled product accumulated from the 32P-C-labeled pre-let-7 RNA. 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, 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 (Hutvagner, 2001).
Next, the products of an in vitro reaction were analyzed by Northern hybridization using three different deoxyoligonucleotide probes. Probe 2 was entirely complementary to mature let-7. Probe 3 was complementary to the first 21 nt of the precursor and therefore only partially complementary to mature let-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 encoding let-7. Finally, probe 4 was complementary to the side of the stem of the precursor opposite the portion encoding let-7. 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-7 itself. 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. Probe 2, which is complementary to let-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. 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 (Hutvagner, 2001).
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, the 5' and 3' ends of both the let-7 generated by the in vitro processing reaction and the let-7 from pupae were analyzed. Treatment with periodate, followed by ß-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. 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. Furthermore, treatment of the CIP-treated RNA with polynucleotide kinase and ATP restored its original mobility, demonstrating that let-7 contains a monophosphate. Because let-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 (Hutvagner, 2001).
The small interfering RNAs (siRNAs) that mediate RNAi also bear a 5' monophosphate and 2'- and 3'-terminal hydroxyls. In Drosophila, siRNA duplexes are produced by the cleavage of long dsRNA by the enzyme Dicer (Bernstein, 2001). 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 (Filippov, 2000) and Dicer. Dicer is the only RNase III domain protein in the publicly available sequence of the Drosophila genome that contains an ATP-binding motif, the DEAD-box RNA helicase domain (Bernstein, 2001). Cleavage of dsRNA by Dicer is strictly ATP-dependent (Bernstein, 2001). Cleavage of pre-let-7 into mature let-7 in Drosophila embryo lysates also required ATP. Taken together, the chemical structure of mature let-7 RNA in vitro and in vivo and the ATP dependence of pre-let-7 processing in vitro strongly implicate Dicer in let-7 maturation. However, it is noted that expression of Dicer protein in Drosophila larvae or pupae has not yet been demonstrated, although the RNAi pathway, which requires Dicer, functions in larvae and pupae (Hutvagner, 2001).
A more stringent test for a role for Dicer in pre-let-7 processing would be to assay let-7 production in flies lacking Dicer protein. However, mutant alleles of Dicer have yet to be identified in Drosophila. As an alternative approach, a recently reported sequence-specific method was used in which cultured mammalian cells were transfected with synthetic 21-nt siRNA duplexes to suppress gene expression. 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 (Hutvagner, 2001).
This method was used 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 to Drosophila Dicer. 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. 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. The level of Dicer mRNA in the Dicer siRNA-treated cells was four- to six-fold lower than in the control samples, whereas actin mRNA levels were unchanged. 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 (Hutvagner, 2001).
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. These products are consistent with the accumulation of the predicted human let-7 precursor RNA (Pasquinelli, 2000) and with a longer form of this precursor containing an extended stem. The mature human let-7 RNA was readily detected in control cells, but not in the cells transfected with the Dicer siRNA duplex, providing additional evidence for a role for Dicer in let-7 maturation. These findings, together with 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. The two pathways must also diverge after the action of Dicer, because siRNA duplexes are generated from long, dsRNA direct mRNA cleavage, whereas the single-stranded stRNA let-7 represses mRNA translation (Hutvagner, 2001).
Recently, Grishok (2001) has shown that the Dicer homolog Dcr-1 is required for both lin-4 and let-7 function in C. elegans. Thus, Dicer is likely to have a broad role in the biogenesis of stRNAs and perhaps other small regulatory RNAs. Furthermore, mutations in the Arabidopsis homolog of Dicer, SIN-1/CARPEL FACTORY (SIN1/CAF), have dramatic developmental consequences (A. Ray, 1996; S. Ray, 1996; Jacobsen, 1999). Perhaps SIN1/CAF protein in plants, like Dicer in bilateral animals, processes structured RNA precursors into small RNAs that regulate development (Hutvagner, 2001).
Pre-let-7 is processed asymmetrically to yield only let-7. It is not yet known 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. The proposed let-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 (Hutvagner, 2001).
microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqsf00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).
To examine the functional connection between the Dicer-1-Loqs complex and pre-miRNA processing, whether depletion of Dicer-1 or Loqs has any effect on the production of mature miRNA from the precursor was investigated. First whether cytoplasmic lysates of S2 cells are capable of processing synthetic Drosophila let-7 precursor RNA into functional mature let-7 was investigated. In this experiment, the synthetic let-7 precursor RNA was converted to mature let-7 in S2 cytoplasmic lysates, as is the case in embryo lysates. In an in vitro RNAi assay, target RNA harboring a sequence perfectly complementary to mature let-7 was cleaved efficiently within the let-7 complementary sequence, thus showing production of functional let-7 in S2 cell lysates. Cytoplasmic lysates from Dicer-1- or Loqs-depleted cells were then subjected to the pre-let-7 processing assay. Both Dicer-1 and Loqs depletion led to reductions of mature let-7 compared with controls, showing that both Dicer-1 and Loqs function in pre-miRNA processing (Saito, 2005).
RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC 1(RNA-induced silencing complex 1). Four Drosophila components (short interfering RNAs, Argonaute 2, VIG and FXR3) of a RISC enzyme have been identified that degrade specific mRNAs in response to a double-stranded-RNA trigger. Tudor-SN (tudor staphylococcal nuclease) -- a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain -- is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi (Caudy, 2003).
Exposure of cells to double-stranded RNA (dsRNA) can elicit various types of sequence-specific gene silencing. A signature of these silencing events is the involvement of small RNAs of approximately 22-25 nucleotides (nt) that guide the selection of silencing targets. These short interfering RNAs (siRNAs) or microRNAs (miRNAs) are generated by the processing of silencing triggers by an RNaseIII family nuclease, Dicer. Small RNAs join multicomponent ribonucleoprotein (RNP) complexes, known generically as RISCs, which enforce silencing (Caudy, 2003 and references therein).
Both to address the nature of the RNAi effector machinery in detail, and to examine the relationship between the different effector mechanisms of RNAi, a RISC complex has been biochemically purified from Drosophila that degrades its mRNA target, and its protein and RNA components have been identified. In multiple, independent purifications of RISC, along with previously characterized proteins, a potentially novel component corresponding to a Drosophila candidate gene, CG7008, has been identified. This evolutionarily conserved 103 kDa protein contains five repeats of a staphylococcal/micrococcal nuclease domain. Four of these repeats are intact, whereas the fifth repeat is fused at its amino terminus to a tudor domain, which has been implicated in the binding of modified amino acids. On the basis of this characteristic domain structure, the protein was named Tudor-SN, for tudor staphylococcal nuclease. Through each purification step, Tudor-SN co-fractionates with known RISC components (Caudy, 2003).
Orthologs of Tudor-SN are found in plants (Arabidopsis9), C. elegans, mammals and Schizosaccharomyces pombe, but not in Saccharomyces cerevisiae. To investigate whether a role for Tudor-SN orthologs in RNAi is evolutionarily conserved, biochemical fractionation of extracts were carried out from C. elegans and mammalian cells (Caudy, 2003).
Cytosolic extracts were prepared from synchronized cultures of wild-type C. elegans. As in Drosophila, a large fraction of the miRNA population can be extracted from the ribosomes. Size fractionation of extracts derived from adult animals has revealed that miRNAs eluted from the column in two peaks, representing 500 kDa and 250 kDa complexes, similar to what has been observed previously in extracts from Drosophila S2 cells. Three different miRNAs -- lin-4, let-7 and mir-52 -- behaved identically in this assay. By contrast, size fractionation of C. elegans egg extract, and examination of complexes containing mir-40 and mir-52, suggests the presence of only the 500 kDa complex. Thus, it seems that miRNAs in C. elegans can inhabit multiple, distinct RNP complexes, and that the partitioning of miRNAs between these complexes may depend on both the identity of the miRNA and the developmental stage of the organism (Caudy, 2003).
RISC complexes in C. elegans have not previously been characterized. Therefore, whether Drosophila RISC components co-fractionate with miRNAs in C. elegans extracts was probed. Antibodies were raised to F56D12.5 (VIG-1), the worm homolog of Drosophila VIG, and F10G7.2 (TSN-1), the worm ortholog of Tudor-SN. Both VIG-1 and TSN-1 were enriched in the fractions that contained 250 kDa miRNA. By contrast, VIG-1 or TSN-1 did not appear in fractions containing 500 kDa miRNA complex (Caudy, 2003).
To test whether putative RISC components are present in the same complex, antibodies were used to immunoprecipitate individual components. In Drosophila, antibodies to either FXR or VIG co-immunoprecipitated Argonaute 2 (Ago-2), as was predicted by findings using epitope-tagged versions of these proteins. Similar amounts of Ago-2 were also co-immunoprecipitated using antibodies directed against Tudor-SN. Furthermore, antibodies directed against FXR and VIG co-immunoprecipitate Tudor-SN, and VIG and Tudor-SN antisera conversely recover FXR, indicating that all four proteins are present in a single complex. In C. elegans, VIG-1-specific antibodies can co-immunoprecipitate TSN-1, indicating a similar association of the C. elegans orthologs of Drosophila RISC proteins (Caudy, 2003).
In naive mammalian cells, it was difficult to detect interactions between orthologs of Drosophila RISC components. However, the formation of a complex containing these proteins was induced if an RNAi response was first triggered by transfection with siRNAs. Specifically, interactions were shown between an Argonaute family protein, AGO2 (human GERp95/EIF2C2/AGO2), the fragile X mental retardation protein (FMRP) and the mammalian Tudor-SN homolog, p100. Notably, complex formation occurred without changes in the expression levels of individual RISC components, indicating that association of pre-existing proteins is nucleated when a siRNA becomes available in the cell (Caudy, 2003).
RISC is an RNP complex that can contain either siRNAs or miRNAs. Consistent with their roles as components of RISC, both miRNAs and siRNAs can be co-immunoprecipitated from Drosophila cells using antisera that recognize FXR, VIG and Tudor-SN. Similarly, in C. elegans, TSN-1 and VIG-1 immunoprecipitates contain let-7 RNA. In addition, the mammalian let-7 miRNA was found in immunoprecipitates of p100, and in parallel the previously demonstrated association between miRNAs and AGO2 was confirmed. miRNAs were also detected in association with members of the fragile X family in mammalian cells, including FMRP, FXR1 and FXR2. Considered together, these results point to a common architecture for RISC in animals as a complex that contains a small RNA (miRNA or siRNA) and protein components that include an Argonaute family member, VIG, Tudor-SN and, at least in Drosophila and mammals, a fragile X family member (Caudy, 2003).
The mammalian homolog of Tudor-SN, known as p100, has been implicated as a co-activator for an Epstein-Barr virus transcription factor, EBNA-2. An exclusively nuclear localization of Tudor-SN would be inconsistent with a role in RNAi, since many studies of RNAi in C. elegans, Drosophila, Neurospora and mammals have shown that post-transcriptional gene silencing by RNAi occurs largely in the cytoplasm. In both Drosophila and mammalian cells, Tudor-SN/p100 is present predominantly in cytoplasmic fractions. Examination of Tudor-SN immunoreactivity also shows a predominantly cytoplasmic localization. In C. elegans, TSN-1 is found in significant amounts both in the nucleus and in the cytosol (Caudy, 2003).
RISC is a nuclease that catalyses endonucleolytic cleavage of substrates, as directed by the associated siRNA. In many cases, siRNAs also trigger mRNA degradation, and the results of biochemical purification are consistent with an association between RISC and a nuclease that catalyses complete destruction of targeted mRNAs. To assess the possibility that Tudor-SN might contribute to catalysis by RISC, its intrinsic nuclease activity was examined. All five staphylococcal nuclease domains of Tudor-SN contain mutations that alter the canonical active site, as derived from comparisons of bacterial family members and from structural data. A large panel of mutations has been made in staphyloccocal nuclease, some of which are similar to those that alter Tudor-SN domains away from the active-site consensus. Generally, these mutations lower the reaction rate but do not abolish catalysis (Caudy, 2003).
Recombinant Drosophila Tudor-SN was expressed and purified from Escherichia coli. Nuclease activity precisely co-fractionates with the recombinant protein, and monospecific carboxy-terminal Tudor-SN anti-peptide antibodies selectively deplete activity from purified Tudor-SN preparations. In these depletion experiments, nuclease activity was recovered on antibody-sepharose complexes. Micrococcal nucleases show broad substrate specificity, cutting both RNA and DNA. Similarly, Tudor-SN can cleave both RNA and DNA substrates (Caudy, 2003).
3',5'-Deoxythymidine bisphosphate (pdTp), known to be a specific competitive inhibitor of staphylococcal nucleases, inhibits Tudor-SN at 100 µM concentrations, whereas dTp (3'-deoxythymidine monophosphate) does not inhibit Tudor-SN activity. Importantly, RISC activity is also inhibited by pdTp. These data are consistent with the possibility that Tudor-SN contributes at least some of the nuclease activity observed in RNAi effector complexes, but are also consistent with other interpretations (Caudy, 2003).
To examine the role of Tudor-SN in dsRNA-mediated silencing in vivo, use was made of a reporter system in C. elegans. A lacZ-lin-41 fusion transgene expresses LacZ in the seam cells under the translational control of let-7. In wild-type animals, LacZ staining in the seam cells can be observed from the L1 stage through to L4. The staining is absent in adult animals as a consequence of let-7-mediated repression. As expected, suppression of alg-1 and dcr-1 by RNAi resulted in persistence of LacZ staining in adults. Next, RNAi was used to analyse the involvement of VIG-1 and TSN-1 in let-7 function. RNAi against either VIG-1 or TSN-1 results in persistent expression of the LacZ reporter in the seam cells of adult animals. This shows that VIG-1 and TSN-1 are required for proper function of the let-7 miRNA in vivo. By contrast, after RNAi against VIG-1, TSN-1 and DCR-1, no effect on RNAi efficiency was seed. The effects observed after RNAi against VIG-1, TSN-1 and DCR-1 probably reflect a partial reduction of function, since other phenotypes associated with let-7 loss of function (vulva and alae defects) are not observed (Caudy, 2003).
These data strongly indicate that Tudor-SN is a bona fide RISC component. This is reflected by the co-purification of Tudor-SN and RISC in Drosophila, C. elegans and mammalian cells. However, it remains open to question whether Tudor-SN is a catalytic engine of RNAi. Despite the aforementioned data, there are potential inconsistencies. (1) Purified, recombinant Tudor-SN is non-sequence-specific, in contrast to RISC, which shows a high degree of selectivity for its mRNA targets. (2)Tudor-SN will cleave both RNA and DNA, whereas no DNase activity is detected in RISC. (3) Several investigators have detected specific cleavage of mRNAs within the siRNA-mRNA hybrid, and this is difficult to rationalize with the known activities of Tudor-SN and related enzymes. It is certainly consistent with the biochemical data to suppose that RISC contains multiple nucleases, only one of which (the putative Slicer) can catalyse site-specific mRNA cleavage. In this scenario, Tudor-SN might act to degrade the remainder of the mRNA. In accord with this idea, targeting of an mRNA by a single siRNA often results in complete degradation of the mRNA. Alternatively, it is possible that Tudor-SN does not have a catalytic role in the RISC complex. Indeed, pdTp is a competitive inhibitor that engages the potential nucleic-acid-binding domains of Tudor-SN. Thus, inhibition of RISC by pdTp may reflect a block in the ability of Tudor-SN to engage RNAs, possibly including the mRNA target, in the context of the RISC complex. Answers to these questions will come only from understanding RISC in sufficient detail to allow reconstitution of its native activity from purified components such that the individual contributions of each to the varied roles of the RNAi effector machinery can be studied in detail (Caudy, 2003).
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