Nine human neurodegenerative diseases are due to expansion of a CAG repeat- encoding glutamine within the open reading frame of the respective genes. Polyglutamine (polyQ) expansion confers dominant toxicity, resulting in neuronal degeneration. MicroRNAs (miRNAs) have been shown to modulate programmed cell death during development. To address whether miRNA pathways play a role in neurodegeneration, whether genes critical for miRNA processing modulate toxicity induced by the spinocerebellar ataxia type 3 (SCA3) protein was tested. These studies reveal a striking enhancement of polyQ toxicity upon reduction of miRNA processing in Drosophila and human cells. In parallel genetic screens, the miRNA bantam (ban) was identified as a potent modulator of both polyQ and tau toxicity in flies. These studies suggest that ban functions downstream of toxicity of the SCA3 protein, to prevent degeneration. These findings indicate that miRNA pathways dramatically modulate polyQ- and tau-induced neurodegeneration, providing the foundation for new insight into therapeutics (Bilen, 2006).
This study reveals a striking role for miRNA-regulated pathways in modulation of polyQ toxicity in both flies and human cells. Notably, reduction of genes that affect miRNA processing, but not siRNA processing, in Drosophila modulates polyQ degeneration, underscoring the specificity to miRNA pathways. In Drosophila, one of these miRNAs is ban, which modulates cell survival upon polyQ- and tau-induced neurodegeneration. Moreover, reduction of miRNA processing in human cells also strikingly enhances polyQ toxicity, indicating that miRNAs also play a protective role in human cells. These data suggest that ban and potentially additional miRNAs are involved in mitigating polyQ- and tau-induced neurodegeneration (Bilen, 2006).
Given the role of miRNAs in modulation of developmental programmed cell death, this study tested whether miRNA pathways modulated neurodegeneration. This was addressed by reducing miRNA processing in flies and in human cells in the presence of pathogenic polyQ protein. In flies, loss of dcr-1 or R3D1 had striking effects to enhance SCA3-induced neurodegeneration. In contrast, loss of dcr-2, which is specific to siRNA pathways, had little effect in the situation tested. These studies indicate that miRNA-regulated activities, and not siRNA-regulated activities, are critical to neurodegeneration in vivo. In human cells, reduction of dicer activity also dramatically enhances cell toxicity induced by pathogenic Ataxin-3. Although, in vertebrate cells in culture, dicer activity affects both miRNA- and siRNA-regulated activities, siRNA-dependent activities like heterochromatic silencing do not become disrupted until later time periods. The effect of reducing dicer in human cells could be rescued in part by complementing the treated cells with a fraction containing miRNAs, indicating that the enhanced cell loss was likely due to reduction in one or more miRNAs. Since miRNAs could affect many cellular processes, it was confirmed that enhanced degeneration with polyQ is unlikely due to sensitizing cells to programmed cell death but rather resembles normal polyQ degeneration; identification of miRNAs and target genes will further define the pathways involved. There are hundreds of miRNAs in humans, with a subset expressed in both human brain and HeLa cells. Focus on these common miRNAs, coupled with the demonstrated ability to rescue polyQ toxicity associated with dicer deprivation, promises to reveal genes with a critical role in neuroprotection from polyQ-induced degeneration. These findings were extended with ban and genes of the miRNA pathway beyond polyQ toxicity to modulation of tau; these findings suggest a broader role for miRNA regulated pathways in neuroprotection (Bilen, 2006).
A genetic modifier screen in Drosophila revealed that one miRNA that functions to modulate Ataxin-3 degeneration is ban. ban is a critical regulator in that both loss of activity and upregulation modulated degeneration. ban mitigated not only degeneration induced by polyQ protein but also by tau, an unrelated neurodegenerative disease protein. Although ban mitigates programmed cell death through hid, these studies indicate that hid is not involved in modulation of degeneration induced by pathogenic Ataxin-3 (Bilen, 2006).
These studies suggest that ban modulates survival of cells to pathogenic polyQ protein downstream of protein accumulation, cellular stress response, and inherent protein toxicity. In the presence or absence of added ban, the pathogenic polyQ protein was present at similar levels and elicited a similar stress response. This indicates that ban regulates progression of degeneration downstream of these events. Taken together, these results suggest that ban may modulate the survival of cells. Indeed, ban may modulate cell survival in multiple situations: after initiation of programmed cell death, as well as in response to neurodegenerative disease proteins, including polyQ and tau. These studies also suggest a role for additional miRNAs, due to the stronger enhancement of polyQ degeneration upon reduction of miRNA processing compared to reduction of ban function alone. This suggests that miRNAs in addition to ban in Drosophila likely play a role in regulating neurodegeneration. Moreover, although loss of miRNA processing results in an overall enhancement, specific miRNAs may be protective, whereas others promote degeneration. It is also possible that proteins involved in miRNA processing could themselves be targets of polyQ toxicity (Bilen, 2006).
These findings expand the role of miRNA function from programmed cell death pathways, developmental processes, and cancer to suggest a striking role in protection from cellular degeneration associated with human neurodegenerative disease proteins. Further identification of the miRNAs and their targets will reveal new insight into mechanisms and therapeutics for the treatment of polyQ, tau-associated, and potentially other neurodegenerative diseases (Bilen, 2006).
The correlation between elevated sensor (expressing GFP under control of the tubulin promoter and two copies of the bantam target in the 3′UTR) expression and the zone of nonproliferating cells adjacent to the dorsoventral boundary (ZNC) suggests that Wingless might control cell proliferation in the ZNC by reducing bantam miRNA levels. To test this, use was made of a dominant-negative form of the Wingless receptor DFz2 to locally reduce Wingless activity. Expression of DFz2-GPI under engrailed-Gal4 control reduces bantam sensor levels in the ZNC of the posterior compartment, indicating that bantam miRNA expression increases when Wingless signaling is impaired. Consequently, cells in the posterior ZNC continue to undergo DNA synthesis and are labeled by BrdU incorporation. Comparable results were obtained by overexpression of the Wingless-pathway inhibitor naked. These observations indicate that Wnt signaling contributes to bantam miRNA expression to exert developmental control over cell proliferation in the ZNC. It is noted that the entire posterior compartment is small under these conditions because Wingless is required earlier to promote overall growth of the wing pouch, in addition to its later role in specifying the ZNC. A second area of reduced bantam sensor expression was noted immediately anterior to the AP boundary, where Hedgehog signaling has been shown to induce cell proliferation. These observations provide a link between signaling proteins that serve as morphogens to control spatial pattern and bantam, a regulator of cell proliferation (Brennecke, 2003).
The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Hippo signaling restricts tissue size by promoting apoptosis and cell-cycle arrest, and animals carrying clones of cells mutant for hippo develop severely overgrown adult structures. The Hippo pathway is thought to exert its effects by modulating gene expression through the phosphorylation of the transcriptional coactivator Yorkie. However, how Yorkie regulates growth, and thus the identities of downstream target genes that mediate the effects of Hippo signaling, are largely unknown. This study reports that the bantam microRNA is a downstream target of the Hippo signaling pathway. In common with Hippo signaling, the bantam microRNA controls tissue size by regulating cell proliferation and apoptosis. hippo mutant cells had elevated levels of bantam activity; and bantam is required for Yorkie-driven overgrowth. Additionally, overexpression of bantam is sufficient to rescue growth defects of yorkie mutant cells and to suppress the cell death induced by Hippo hyperactivation. Hippo regulates bantam independently of cyclin E and diap1, two other Hippo targets, and overexpression of bantam mimics overgrowth phenotypes of hippo mutant cells. These data indicate that bantam is an essential target of the Hippo signaling pathway to regulate cell proliferation, cell death, and thus tissue size (Nolo, 2006).
To test whether the activity of the bantam miRNA is regulated by Hpo signaling, use was made of a GFP bantam sensor that reports the spatial activity of bantam. This bantam sensor expresses GFP under the control of a ubiquitously active tubulin promoter and has two perfect bantam target sites in its 3′ UTR. When present, the bantam miRNA reduces GFP expression through its RNAi effect. The expression pattern of GFP is thus a negative image of the activity pattern of the bantam miRNA. In third-instar wing imaginal discs, the bantam sensor is expressed in a complex pattern with higher levels along the presumptive wing margin, in the anterior compartment along the anteroposterior compartment boundary, and in several patches in the thorax region. Overexpression of the bantam miRNA in the developing wing eliminated the GFP expression of the bantam sensor in the corresponding region, demonstrating that the expression of GFP is indeed under the control of bantam. In developing eye discs, the bantam sensor is also broadly expressed, with higher levels in differentiating photoreceptor cells. As in wing discs, overexpression of bantam downregulated GFP expression in eye discs. The bantam sensor thus reflects the activity of the bantam miRNA in eye and wing discs (Nolo, 2006).
To address whether Hpo signaling regulates the activity of the bantam miRNA, GFP expression of the bantam sensor was monitored in imaginal discs that had defects in Hpo signaling. It was found that hpo or wts mutant cells had lower levels of bantam-sensor-driven GFP expression throughout the mutant clones. Significantly, hpo and wts mutant clones showed lower levels of GFP in multiple tissues, including the wing, antenna, and eye imaginal discs. In eye imaginal discs, wts clones affected the bantam sensor anterior to the morphogenetic furrow, where cells are still uncommitted as well as posterior to the furrow in differentiating photoreceptor cells. In all cases, the regulation of the bantam sensor was cell autonomous. In addition, wing imaginal discs that overexpressed Yki had lower levels of bantam sensor expression in the entire region of Yki overexpression. In summary, it is concluded that Hpo signaling generally regulates bantam expression in multiple imaginal discs and cell types (Nolo, 2006).
A model is postulated in which bantam is an essential target of the Hpo signaling pathway to regulate cell proliferation, cell death, and thus tissue size. This model is based on several observations. First, it was found that bantam is regulated by Hpo signaling broadly and in various tissues. This regulation is a specific downstream effect of Hpo signaling and is not simply the consequence of the cell proliferation induced in hpo mutant cells. Second, bantam is required for Yki to drive tissue overgrowth, because removal of bantam suppresses the overgrowth phenotypes caused by overexpression of Yki in the retina. Third, overexpression of bantam rescues the cell death induced by overexpressed Hpo and significantly rescues growth defects of yki mutant cells. And fourth, bantam overexpression mimics the phenotypes of hypomorphic hpo mutations. Taken together, these data support a model in which bantam is an important downstream target of the Hpo pathway (Nolo, 2006).
The finding that Hpo signaling regulates the expression of bantam raises the question of how important this effect is for Hpo signaling to control tissue size. Removal of bantam suppresses the induction of extra interommatidial cells in the retina by Yki overexpression but does not cause a general elimination of retinal cells in a wild-type background. These data indicate that the regulation of bantam is an essential downstream effect of Hpo signaling to regulate tissue size. However, loss of bantam only partially suppresses the effects of Yki overexpression, indicating that Yki regulates other targets in addition to bantam. Hpo was found to regulate bantam independently of cyclin E and diap1, two other genes known to be regulated by Hpo signaling. bantam is thus not a component of the Hpo signal transduction pathway itself, but is one of several downstream target genes. Yki must have targets in addition to bantam, cyclin E, and diap1, because overexpression of bantam, Cyclin E, and DIAP1 together did not induce the amount of overgrowth caused by Yki overexpression in wing discs. Nevertheless, overexpression of bantam alone caused phenotypes resembling hypomorphic situations for Hpo signaling, indicating that bantam is a critical mediator of Hpo function. Whether the regulation of bantam by a Yki-containing transcription factor complex is direct remains to be determined. However, the fact that Hpo regulates bantam cell autonomously and in multiple tissues is consistent with such a model (Nolo, 2006).
bantam expression is spatially modulated, and patterning signals such as Wg and Dpp also regulate the expression of bantam to generate its expression pattern. These patterning signals regulate specific aspects of the bantam expression pattern, and they have different effects on cell proliferation as well as bantam activity in different regions in various imaginal discs. In contrast, hpo mutant cells upregulate bantam activity independently of cell type and in multiple imaginal discs, indicating an intimate relationship. Hpo is thus a more general and ubiquitous regulator of bantam expression in imaginal discs. An important question that remains to be answered is how these patterning signals regulate tissue growth and bantam expression and whether they regulate bantam expression directly and independently of Hpo signaling or through the regulation of Hpo activity (Nolo, 2006).
Surprisingly, just the opposite of hpo mutant cells, TSC1 mutant cells had lower levels of bantam activity although these cells overgrow, indicating that TSC1 mutant cells induce growth independently of bantam. Neither Myc, Ras, nor Cyclin D-Cdk4 expression induced bantam, although they induce cell growth and proliferation. bantam is thus not simply a part of the cell-intrinsic machinery that executes cell growth and division but rather acts as an upstream component to instruct cells to proliferate. In summary, although Hpo is a key regulator of bantam expression, bantam is also regulated by other pathways potentially integrating the effects of several growth-regulatory and patterning pathways (Nolo, 2006).
miRNAs and their target genes often show mutually exclusive expression patterns, and miRNAs induced during differentiation tend to target messages that were abundant in the previous developmental stage. miRNAs may thus provide a rapid and effective means to suppress expression of residual, unwanted mRNAs while the transcriptional program in a cell is changing. Hpo signaling is involved in regulating cell proliferation and apoptosis in developing imaginal discs. Cell lineages and cell proliferation show significant plasticity in growing imaginal discs, which can rapidly respond to surgical ablation or genetic insults by regenerating missing (eliminated) cells or by ablating unwanted (extra) cells. This adjustment of cell proliferation and apoptosis requires a mechanism that can rapidly change the growth properties of a cell. Yki appears to regulate cell number on the one hand by inducing the expression of positive regulators of cell proliferation and cell survival and on the other hand by inducing the expression of bantam, which posttranscriptionally suppresses the expression of proteins that inhibit cell proliferation and induce apoptosis. An example of such cooperative action of Yki and bantam is the regulation of Hid: Yki suppresses the expression of hid, but also induces bantam, which then suppresses the translation of hid mRNAs that may still be present in a cell. The induction of bantam by Yki may also accelerate the repression of negative growth regulators, thereby enabling a cell to more quickly and robustly adjust its rate of cell proliferation. It will be interesting to elucidate how bantam regulates growth and how its growth targets are integrated with other targets of Hpo signaling (Nolo, 2006).
The Hippo signaling pathway acts upon the Yorkie transcriptional activator to control tissue growth in Drosophila. Activated Yorkie drives growth by stimulating cell proliferation and inhibiting apoptosis, but how it achieves this is not understood. Yorkie is known to activate Cyclin E (CycE) and the apoptosis inhibitor DIAP1. However, overexpression of these targets is not sufficient to cause tissue overgrowth. This study shows that Yorkie also activates expression of the bantam microRNA, a known regulator of both proliferation and apoptosis. bantam overexpression mimics Yorkie activation while loss of bantam function slows the rate of cell proliferation. bantam is necessary for Yorkie-induced overproliferation and bantam overexpression is sufficient to rescue survival and proliferation of yorkie mutant cells. Finally, bantam levels are shown to be regulated during both developmentally programmed proliferation arrest and apoptosis. In summary, the results show that the Hippo pathway regulates expression of bantam to control tissue growth in Drosophila (Thompson, 2006).
The Hippo pathway is unique in its direct and dedicated role in the intrinsic program of growth in proliferating tissues. The potency of the Hippo pathway in driving tissue growth appears to reside in its ability to coordinately stimulate cell proliferation and suppress apoptosis. A key goal is to understand how this coordinate control is achieved. The results show that the bantam microRNA, a known regulator of both cell proliferation and apoptosis, is a critical target of the Hippo pathway. Activated Yki is necessary and sufficient to induce bantam expression and to stimulate cell survival and proliferation. bantam appears to be a key target of Yki because loss of Yki can be rescued by overexpression of bantam. Finally, bantam clearly has an important role in both normal growth and Yki-driven overgrowth because loss of bantam strongly reduces the rate of cell proliferation in either case. Although the bantam microRNA appears not to be conserved in vertebrates, it is possible that other microRNAs play a functionally equivalent role as effectors of the Hippo pathway. Recent work has identified human microRNAs involved in this pathway (Thompson, 2006).
Two lines of evidence indicate that bantam is not the only relevant target of the Hippo pathway. Firstly, loss of bantam does not completely mimic loss of Yki in every respect, because bantam mutant cells do not undergo apoptosis. This difference is likely to reflect the contribution of the Yki target DIAP1, whose absence is known to trigger apoptosis. Secondly, Yki retains some ability to stimulate cell proliferation even in the absence of bantam. Again, this activity may reflect the role of other Yki targets, including CycE, in driving cell proliferation. Thus, the results favor the view that bantam acts in a highly cooperative way with other Yki target genes to mediate the effects of the Hippo pathway on cell proliferation and apoptosis (Thompson, 2006).
The expression of bantam during normal development shows a striking pattern of regulation; it is expressed in proliferating cells but not in quiescent cells or, as has been shown in this work, in certain cells destined for apoptosis. These findings indicate that regulation of bantam is a key feature of the normal program of tissue growth. Previous work has shown that high levels of the Wingless (Wnt) morphogen represses bantam as cells arrest proliferation at the presumptive wing margin. Since the results show that the Hippo pathway regulates bantam, the pattern of bantam expression may reflect regulation of Hippo pathway activity by positional signals. Thus, positional signals could determine the behavior of cells along the spectrum from rapid proliferation to apoptosis simply by controlling the Hippo pathway. Alternatively, positional signals and the Hippo pathway may act independently, with the bantam locus being a regulatory nexus that integrates information from a number of different signaling pathways (Thompson, 2006).
The final finding is that the Hippo pathway also influences epithelial morphogenesis. This function appears to be independent of its role in controlling cell survival and proliferation and does not involve bantam. Interestingly, expression of Yki and Hippo have reciprocal effects on the epithelium, with overexpressed Yki driving apical bulging and overexpressed Hippo causing basal outfolding. In both cases, the cells remain epithelial, indicating that the Hippo pathway controls cell shape without affecting epithelial polarity or integrity. These observations are consistent with previous reports that clones of cells with elevated pathway activity (i.e. mutant for Hippo or other negatively acting components) have a rounded appearance, indicating altered cell affinities, and that mutation of warts also causes apical bulging of epithelia that is attributed to an expanded apical membrane domain. Why cells use the same pathway to control survival, proliferation, and shape remains an intriguing question (Thompson, 2006).
A fuller understanding of the network connecting the Hippo pathway with the basic machinery controlling survival, proliferation, and morphology will be needed to understand how size regulation is connected to pattern formation during normal development. This work allows sketching of the outline of one facet of this network, with the Yki targets bantam, CycE, and DIAP1 cooperating to control survival and proliferation (Thompson, 2006).
Studies on the Myc and E2F oncogenes in vertebrates have shown that strong proliferative stimuli induce apoptosis. Cell proliferation results only when apoptosis is simultaneously prevented. Overexpression of E2F with its cofactor DP causes apoptosis in the Drosophila wing disc, and net cell proliferation results only when apoptosis is blocked by coexpression of the caspase inhibitor P35. In contrast, stimulation of growth by bantam overexpression is not associated with an increase in apoptosis. This raises the possibility that bantam might stimulate cell proliferation and simultaneously suppress apoptosis. It was asked whether bantam could suppress proliferation-induced apoptosis, caused by E2F and DP. Cells expressing E2F and DP under ptc-GAL4 overproliferate, indicated by increased nuclear density in apical optical sections. In basal optical sections, elevated levels of activated caspase 3 are seen. Many of these cells drop out of the epithelial layer and have pyknotic nuclei, indicative of apoptosis. Coexpression of bantam enhances the overproliferation phenotype, indicated by the broader region of high nuclear density and reduces the levels of activated caspase. Fewer cells show pyknotic nuclei, although many cells drop out of the epithelial layer, indicating that they are not entirely healthy. Even the modest level of bantam overexpression produced by EP(3)3622 is sufficient to suppress apoptosis induced by E2F and DP overexpression (Brennecke, 2003).
To identify targets of the bantam miRNA, a computational method was developed based on the known C. elegans miRNA-target pairs and a general understanding of RNA-RNA interactions. A target search using bantam miRNA revealed three independent targets in the 3′UTR of the apoptosis-inducing gene hid. By visual inspection of the 3′UTR of hid, two additional sequences complementary to the bantam miRNA were identified. All five target sites are highly conserved in the predicted hid 3′UTR of D. pseudoobscura. The bantam precursor hairpin from D. pseudoobscura is identical to that from D. melanogaster, except for one base in the terminal loop that is not in the miRNA product. The conservation of these sequences suggests a conserved functional relationship between bantam and hid (Brennecke, 2003).
To assess the function of the predicted bantam target sites, a tubulin-EGFP sensor transgene was produced using the 3′UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor. In addition, the hid UTR sensor is downregulated when EP(3)3622 is overexpressed under ptc-Gal4 control, indicating that the hid UTR confers bantam-dependent regulation on the transgene. The hid UTR sensor was compared with a version from which bantam target sites one and four were deleted. The mutated sensor with the three gapped bantam sites showed a similar pattern to the complete hid UTR sensor, but was downregulated less strongly by endogenous bantam. Overexpressed bantam reduced its expression, but the difference in magnitude was less than for the intact hid UTR sensor with five sites. These observations indicate that the gapped sites are functional in mediating bantam induced repression, but show that five sites mediate stronger repression than three sites. Cooperativity among multiple sites has also been reported for siRNA-mediated translational repression (Brennecke, 2003).
Having shown that bantam can block expression of a transgene containing the hid 3′UTR, it was asked whether bantam regulates the endogenous hid gene. hid was expressed from EP(3)30060 under ptc-Gal4 control, either alone or together with EP(3)3622. Hid protein levels were reduced by coexpression with, but hid transcript levels were comparable. This indicates that Hid protein expression is repressed by bantam, most likely by blocking translation of the hid mRNA. The ability of bantam to suppress the apoptosis-inducing effects of hid was examined. hid expression induces apoptosis, visualized by caspase 3 activation. This is suppressed by coexpression of bantam. These observations show that bantam effectively suppresses hid-induced apoptosis. The ability of bantam to suppress proliferation-induced apoptosis may reflect its ability to block Hid expression, though the possibility of other indirect effects cannot be excluded (Brennecke, 2003).
Induction of cell death in postmitotic cells of the eye imaginal disc by GMR-hid, GMR-hid(Ala5) and GMR-reaper transgenes leads to a small, rough eye phenotype. Eye size is largely restored by coexpression of bantam using GMR-Gal4 to direct expression of EP(3)3622, though suppression of the GMR-hid and GMR-hid(Ala5) phenotypes is much better. Ommatidial structure is largely restored in the GMR-hid eyes, but not in the GMR-reaper eyes, suggesting a more specific suppression of hid activity. Hid(Ala5) has the 5 consensus ERK phosphorylation sites mutated to alanine and cannot be suppressed by activation of the ERK MAPK pathway. The observation that bantam coexpression blocks the activity of Hid(Ala5) excludes an indirect effect mediated by regulation of the MAPK pathway. To explore the question of specificity further, the effects of removing one copy of the endogenous bantam gene in these three backgrounds was compared. This had a minor effect on the severity of the GMR-reaper, but clearly enhanced the severity of the GMR-hid and GMR-hid(Ala5) phenotypes. This suggests that endogenous expression of the bantam gene in the developing eye imaginal disc contributes to controlling the level of hid-induced apoptosis, which is normally involved in reducing cell number in the pupal eye disc (Brennecke, 2003).
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).
Pre-miR-bantam was used as a substrate for pre-miRNA processing assays. It has been shown that S2 cell extracts contain primary-miRNA processing activity that cleaves pri-miRNA into an approximately 60- to 70-bp pre-miRNA precursor. This processing is known to occur in the nucleus; thus pre-miR-ban was prepared by in vitro processing of pri-miR-ban incubated with S2 nuclear extracts. Uniformly labeled pre-miR-ban was then gel-purified and used as a substrate for analysis of pre-miRNA processing. Incubation of the pre-miRNA with S2 cytoplasmic extracts results in the appearance of a mature 21-nucleotide miR-ban. Then the requirement of Dicer-1 and Loqs in pre-miR-ban processing was examined. Incubation of pre-miRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic extracts results in a marked reduction in mature miRNA levels. In contrast, depletion of Dicer-2 or R2D2 shows no measurable reduction of mature miRNA levels. Then the pre-miRNA processing activity of the purified complexes (both Flag-Dicer-1 and Flag-Loqs complexes) was assayed. That the Flag-Loqs complex contains Dicer-1 was confirmed by immunoblotting. Both Dicer-1 and Loqs complexes are capable of generating maturemiR-ban from pre-miR-ban. Several steps in the RNAi and miRNA pathways are known to require a divalent metal ion. In addition, it is well known that RNase III-type enzymes require divalent metals for cleavage. Flag-Dicer-1 complex was employed and the processing was performed in the presence of magnesium ions or EDTA in a buffer. No pre-miRNA processing activity is detected at 10 mM EDTA. These results demonstrate that the Dicer-1-Loqs complex converts pre-miRNAs into mature miRNAs in a divalent metal ion-dependent manner (Saito, 2005).
To confirm the function of loqs in pre-miRNA processing, cultured Drosophila S2 cells were depleted of loqs mRNA by RNAi. Eight days after incubating S2 cells with dsRNA corresponding to the first 300 nucleotides of the loqs coding sequence, the steady-state levels of pre-miRNA and mature miRNA were determined for miR-277 and bantam. Relative to an unrelated dsRNA control, dsRNA corresponding to dcr-1 caused an approximately 9-fold and approximately 23-fold increase in steady-state pre-miR-277 and bantam levels, respectively, and dsRNA corresponding to loqs caused an approximately 2-fold and approximately 6-fold increase in steady-state pre-miR-277 and bantam levels, respectively. In these experiments, RNAi of dcr-1 more completely depleted Dcr-1 protein than RNAi of loqs reduced Loqs protein. RNAi of dcr-2,r2d2, or drosha did not alter pre-miRNA levels for either miR-277 or bantam, nor did it alter Dcr-1 or Loqs levels. The Drosha/Pasha protein complex functions before pre-miRNA processing, converting primary miRNA (pri-miRNA) to pre-miRNA. Consistent with the idea that Loqs functions with Dcr-1 to convert pre-miRNA to mature miRNA, RNAi of drosha together with loqs alleviates the high pre-miRNA levels observed for RNAi of loqs alone, demonstrating that Loqs acts after Drosha (Forstemann, 2005).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.