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).
The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. This study provides evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and this study shows that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain (Peng, 2009).
The evidence suggests that Hth and Tsh function as partners to carry out two main functions in anterior eye disc cells: They repress the expression of the later-acting retinal determination factors, and they promote cell proliferation. That these functions require hth is supported by both loss-of-function studies as well as gain-of-function studies. For example, hthP2 clones fail to survive anterior to the MF, and Tsh's ability to induce overgrowths when ectopically expressed is abolished in the absence of hth. The involvement of Tsh is supported by gain-of-function experiments and the finding that Hth and Tsh directly interact with each other in vivo. Carrying out loss-of-function genetics for tsh is difficult because this gene is located proximal to the standard Flp recombination targets (FRTs) used to generate mitotic recombination. In addition, the highly related gene tio, which is closely linked to tsh, functions redundantly with tsh in several instances, including some aspects of eye development. Nevertheless, knocking down tsh using RNAi in a tio-null background results in poor survival in the progenitor domain. Taken together, these data provide a compelling argument for Hth + Tsh functioning together to promote cell survival in the anterior eye disc (Peng, 2009).
A functional relationship between Hth and Tsh also exists in other tissues in Drosophila, most notably in both wing and leg imaginal discs, where they are coexpressed in cells that will give rise to the proximal domains of these appendages. In both wings and legs, Tsh has the capacity to regulate hth when expressed in clones, and both tsh and hth have the ability to suppress distal appendage development when misexpressed. However, in these tissues, and unlike the eye disc, Hth + Tsh expression is not correlated with proliferation, which occurs uniformly throughout these discs. Consistently, the expression pattern exhibited by the bantam sensor does not correlate with Hth or Tsh in the leg or wing. The special relationship between proliferation and Hth + Tsh in the eye may be due in part to the Drosophila Pax6 homolog Eyeless (Ey), which is critical for eye identity. Moreover, Ey is found in a complex with Hth in vivo and participates with Hth and Tsh in the repression of retinal determination genes. Thus, it may also be the case that Ey directly participates in the regulation of bantam together with Hth and Yki (Peng, 2009).
Although hthP2 clones fail to survive in the eye progenitor domain, the data demonstrate that hth is not absolutely required for cells in this domain to proliferate. The effects observed on the bantam sensor are consistent with the idea that hth promotes, but is not essential for, cells to proliferate in the eye progenitor domain. In hthP2 clones, bantam sensor levels increased above those normally observed in progenitor cells, but not as high as the levels observed in differentiated photoreceptors. Thus, if the level in photoreceptors represents the complete absence of bantam, these data imply that hth only up-regulates bantam over a basal hth-independent level. Moreover, the levels of the bantam sensor in other tissues, such as the wing disc, rarely approach those observed in photoreceptors, suggesting that most cells have some bantam expression, and that bantam regulators, such as hth, only serve to modulate bantam levels (Peng, 2009).
If eye progenitor cells have the capacity to proliferate in the absence of hth, how important is the proliferation-promoting function of hth? Although normal eyes can develop in animals in which hthP2 clones are generated, this is likely due to the ability of neighboring wild-type cells to compensate in this mosaic situation. In contrast, when wild-type and heterozygous cells are killed (using the EGUF method [ey-Gal4/UAS-flp/GMF-hidwe find that the remaining hthP2 tissue is unable to produce normal-sized eyes. This experiment suggests that the proliferation-promoting functions of hth in the eye progenitor domain are critical for normal eye development, likely by providing a sufficient pool of progenitor cells prior to differentiation (Peng, 2009).
The Hippo pathway has emerged recently as an important regulator of cell proliferation and survival in both vertebrates and invertebrates. In Drosophila, this pathway appears to regulate proliferation in nearly all tissues. For example, wts- clones or Yki+ clones have the capacity to induce overgrowths throughout the body. As Yki and its mammalian ortholog Yap are transcriptional coactivators that do not have their own DNA-binding domain, they are thought to partner with DNA-binding transcription factors to regulate gene expression. Prior to this work, the only transcription factor proposed to work directly with Yki was Sd, a member of the TEAD/TEF family of DNA-binding proteins. However, unlike other components of the Hippo pathway, the available data suggest that Sd plays a more limited role in cell proliferation and survival in Drosophila. In contrast to its essential role in the wing pouch, sd- clones survive well in other tissues, including the region of the wing disc that will give rise to the notum of the fly. Similarly, sd-null clones grow well in the eye progenitor domain. Thus, unlike in the wing pouch, sd is not required for cell survival and proliferation in the eye progenitor domain (Peng, 2009).
In contrast to the survival of sd clones in this domain, hthP2 clones fail to survive in the eye progenitor domain. Thus, analogous to sd in the wing pouch, hth is required for cells to survive and proliferate in the anterior eye imaginal disc. This observation suggests that hth could play an analogous role to sd in this progenitor domain, a view that is supported by the results. This evidence includes (1) Hth can interact with Yki when coexpressed in S2 cells, (2) Hth + Tsh regulate the Yki target bantam, and (3) Hth and Yki are both bound to the same region of the bantam locus in eye discs. Genetically, it was shown that the Hippo pathway is unable to induce overgrowths in the eye progenitor domain without hth, and that Hth + Tsh cannot induce overgrowths in the absence of Yki. These results suggest that Hth + Tsh comprise the DNA-binding transcription factors that function with Yki to regulate proliferation and survival genes, such as bantam. Thus, analogous to Sd in the wing pouch, Hth + Tsh are transcription factors used by the Hippo signaling pathway in eye progenitor cells (Peng, 2009).
The finding that Hth + Tsh play an analogous role in the eye progenitor domain as Sd does in the wing pouch has several implications for how the Hippo pathway is regulated in vivo. For one, the use of different DNA-binding transcription factors to regulate Hippo target genes suggests a previously unknown degree of specificity available to this pathway. Hth, a TALE family homeodomain protein, and Tsh, a Zn finger protein, are likely to bind very different target DNA sequences than Sd, a TEAD/TEF domain DNA-binding factor. Accordingly, it was found that ectopic Hth + Tsh clones in the eye disc do not consistently up-regulate diap1 or expanded, known Sd-Yki targets in the wing disc (Peng, 2009).
These results also imply that the transcriptional regulation of hth, tsh, and sd has the potential to change the output of the Hippo pathway. Because hth and tsh are transcriptionally repressed by signals coming from the MF, these factors are not available to work with the Hippo pathway posterior to the MF. However, loss of Hippo kinase activity can lead to proliferation of differentiated cells posterior to the MF. In these cells, sd is expressed, suggesting that Yki may use this transcription factor in this context. Analogously, loss of Hippo kinase activity can cause overgrowths in the notum as well as in the wing pouch. As sd- clones grow well in the notum, but not in the wing pouch, these data suggest that the notum overgrowths may be mediated by a transcription factor other than Sd. hth clones also survive well in the notum, implying that yet another transcription factor or factors may work with Yki in this tissue. In sum, it is suggested that Yki, and thus the Hippo pathway, may be able to work with multiple transcription factors to regulate target genes. In principle, the use of several transcription factors that are themselves developmentally regulated allows the Hippo pathway to be interpreted in different ways in different contexts (Peng, 2009).
Although the data suggest that the Hippo pathway uses Hth + Tsh to up-regulate bantam, they also suggest that both Hth + Tsh and Yki have additional, independent targets. For example, the loss of Hippo kinase activity leads to the up-regulation of diap1 throughout the eye disc. Because diap1 is not affected when Hth + Tsh are coexpressed, the Hippo pathway has the capacity to regulate some genes independently of Hth + Tsh, even in the eye progenitor domain. Moreover, at least when Yki is ectopically expressed, sd appears to be required in all regions of the eye disc for diap1 activation. Thus, although it has not been shown that sd is required for endogenous diap1 expression in this tissue, these data suggest that Yki may use both Sd and Hth + Tsh to regulate gene expression in the eye disc. In fact, it has been suggested that sd is also a modifier of bantam expression in the eye disc and that sd is required for normal-sized eyes. However, these clones, which used RNAi to knock down Sd, grew well in the eye progenitor domain. In addition, the smaller eyes observed when sd was knocked down could be due to the earlier embryonic expression of the Gal4 driver used in these experiments. In contrast, when generated during larval stages, hth- clones, but not sd- clones, fail to survive in the eye progenitor domain, arguing that, at least post-embryonically, gene regulation by Hth + Tsh, not Sd, is critical for cell survival in this tissue. This conclusion is also supported by the finding that Hth + Tsh can induce proliferation in the absence of sd (Peng, 2009).
As shown previously, Hth + Tsh play a key role in blocking eye differentiation by repressing the retinal determination genes eya and so. The available data do not yet resolve whether this repression works independently of the Hippo pathway. In contrast, the loss of Hippo kinase activity leads to overgrowths without blocking differentiation, arguing that nuclear Yki promotes proliferation without changing cell fate. Consistently, it was found that wts- or Yki+ clones do not alter Elav expression in differentiated photoreceptors. Curiously, however, ectopic expression of Hth + Tsh did not block differentiation in the absence of Yki. Although these data could be interpreted to suggest that Yki is directly required for repressing differentiation, they could alternatively suggest that repression requires cell proliferation. Consistently, Hth + Tsh were also unable to block differentiation in the absence of bantam. These observations raise the possibility that the absence of bantam or yki indirectly inhibits Hth + Tsh's ability to repress differentiation by compromising the proliferation of these cells, although other indirect affects are also possible (Peng, 2009).
Hth + Tsh are also likely to regulate genes in addition to bantam to promote proliferation and survival in the eye progenitor domain. This is most strongly supported by the observation that ectopic expression of bantam only partially rescues the survival of hthP2 clones. In addition, it was found that the overgrowths generated by ectopic expression of Hth + Tsh are only partially suppressed by the coexpression of Hpo, whose overexpression removes Yki from the nucleus. These data suggest that some of the Hth + Tsh targets that mediate growth and survival in the eye progenitor domain are regulated independently of Yki (Peng, 2009).
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).
Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), the miRNA biogenesis pathway was knocked down in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs: clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, circadian cell-specific inhibition of the miRNA biogenesis pathway was exploited followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes (Kadener, 2009).
This study demonstrates a role for miRNAs in the Drosophila central circadian clock. By performing AGO1 immunoprecipitation followed by microarray analysis, a population of mRNAs under miRNA control in fly heads. Among them was the master circadian gene clk. In addition, circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling arrays identified several miRNAs prominently expressed in circadian tissues. In combination with bioinformatics analyses, the two approaches identified 10 candidate miRNAs involved in circadian rhythms. For one miRNA, the developmental regulator bantam, evidence is presented for a direct role in circadian timekeeping. Overexpression of bantam using a circadian cell-specific GAL4 line delays by almost 3 h the circadian clock at the molecular and behavioral levels. Moreover, this miRNA regulates clk. This regulation is achieved through three conserved bantam sites in the 3' UTR of this gene. Two are located downstream from the previously annotated clk mRNA 3' end, and other data indicate that the real clk 3' UTR includes these sites. Genetic experiments in flies demonstrate that the integrity of these three bantam sites is critical for robust circadian rhythmicity. Therefore a miRNA-mRNA pair involved in central circadian timekeeping was identified (Kadener, 2009).
This is one of the few studies to use miRNP IP to identify miRNA-regulated mRNAs, and may be the first from adult fly tissues. The data fit well with those derived from the PicTar algorithm and should allow a comparison of different miRNA target prediction algorithms (Kadener, 2009).
The second approach for studying specific miRNA expression relies on cell type-specific inhibition of miRNA synthesis pathways in vivo followed by RNA analysis on tiling arrays. Although very sensitive in identifying many circadian miRNAs, the strategy probably still fails to identify low abundance miRNAs or miRNAs present in small numbers of circadian cells. However, they should be detectable with the same approach, but after a cell purification or cell sorting step. This sensitivity issue is the reason the broad tim-gal4 driver was used rather than the more restricted pdf-gal4 driver. Tim-gal4 is expressed strongly in all circadian tissues of the fly head, including circadian neurons, eyes, fat body, and antennae. This broad expression also explains the strong effect of TIM-Dcr IR on the AGO1 IP enrichment. Consistent with data indicating that core clock components work similarly in both central (brain) and peripheral tissues, bantam overexpression slows the clock pace in both locations: in the central brain as demonstrated by behavior, and in the periphery as demonstrated by luciferase assays (Kadener, 2009).
Intersecting the Ago1 IP data with the tiling array data from Tim-DroshaIR/PashaIR flies as well as with the published fly head miRNA data led to a selection of 10 candidate circadian miRNAs. Since this analysis only used miRNAs with PicTar target predictions and therefore screened only half of the known miRNA population, 10 is likely to be an underestimate. In contrast, of the 27 miRNAs identified as expressed in circadian cells by the Tim-DroshaIR/PashaIR approach, 23 have mRNAs with PicTar predictions in the Ago IP data. This suggests that 10 is not a gross underestimate (Kadener, 2009).
Some of these 10 miRNAs are likely responsible for the decrease in locomotor activity rhythm strength due to inhibition of the miRNA pathway. It is notable that there are no prior reports of a miRNA contribution to circadian behavior in Drosophila and only a single report in mammals. This may be related to the fact that an effect was only manifest at 29°C and with the addition of the UAS-CYC-VP16 transgene. The failure to observe a phenotype in Tim-DcrIR flies at 25°C may reflect a relatively weak effect of the dicer-1 IR transgene on miRNA expression, consistent with the fact that miRNA biosynthesis is not rate-limiting for miRNA-mediated translational regulation. Nonetheless, it is likely that the lack of a circadian defect in Tim-DcrIR flies is not a consequence of inadequate inhibitory transgene expression. This is because the same strain (Tim-DcrIR) still displays normal rhythms even after increasing the temperature to 29°C. Moreover, Tim-Dcr seems to strongly down-regulate the miRNA pathway, as illustrated by the accumulation of pre-bantam and the substantial change in the AGO1 IP profile (Kadener, 2009).
It is therefore suspected that the additional requirement for UAS-CYC-VP16 reflects more than just an increase in UAS-dcr 1 IR expression. It is possible that the transcription and translation of key circadian core components are tightly connected and may buffer each other. Such a regulatory feature could explain why a major increase in transcription, like that caused by the CYC-VP16 transgene, results in only a modest increase in mRNA abundance and probably an even more modest increase in translated protein. A comparable explanation posits that inhibition of the miRNA pathway by the UAS-dcr 1 IR transgene leads to an increase in the translation of circadian repressors, which could then decrease circadian transcription. The use of UAS-CYC-VP16 as well as 29°C might be required to push the system sufficiently far from equilibrium so that pacemaker regulatory mechanisms can no longer compensate for the change in miRNA levels. This type of regulation fits recent data demonstrating that a Drosophila miRNA can function as a buffering agent against environmental perturbations during development (Li, 2009). In any case, the observed behavioral defects observed in Tim-DcrIR-CYCVP16 flies are likely a consequence of down-regulation of several circadian-relevant miRNAs (Kadener, 2009).
Behavioral, genetic, and biochemical evidence indicates that bantam contributes to clk mRNA translational regulation as well as more generally to circadian pacemaker regulation: bantam is highly expressed in circadian tissues, and overexpression with either tim-gal4 or pdf-gal4 significantly lengthens circadian period. The milder effect of the pdf driver may be due to its lower strength in pacemaker cells relative to tim-gal4 and/or to an additional contribution from non-PDF cells to period determination (Kadener, 2009).
Although the period phenotype could be misleading -- due, for example, to an effect of bantam overexpression on a circadian output pathway -- strains with a completely normal central pacemaker do not manifest altered periods, by definition. Another possibility, that bantam overexpression renders the circadian neurons sick or unhealthy, would be expected to result in weak rhythms or arrhythmicity rather than in strong rhythms with long periods. The central pacemaker is therefore the most parsimonious explanation, especially because of the good correlation between the behavioral and the molecular data; i.e., the tim-luciferase results. Unfortunately, the bantam deletion is embryonic lethal, precluding a straightforward behavioral assay of the null phenotype (Kadener, 2009).
The effect of bantam on clk mRNA translation was aided by the finding that the clk 3' UTR extends >700 bases downstream from its predicted 3' end. This error is attributed to priming by oligo (dT) within an A-rich region present near this annotated 3' end. Consistent with this interpretation, a strongly conserved cleavage and polyadenylation site is present near the end of the clk-lg isoform; no obvious site is in the vicinity of the annotated clk 3' end. In addition, RNA protection data indicate that all fly head clk transcripts extend well beyond the annotated clk 3' end. Taken together with the 3' RACE data, these results demonstrate that the clk 3' UTR is significantly longer than previously indicated. Importantly, two of the three clk 3' UTR bantam-binding sites are located downstream from the annotated 3' end (Kadener, 2009).
These clk 3' UTR bantam sites appear to be major circadian targets of bantam in flies. First, clk mRNA is strongly associated with RISC. Second, bantam is strongly expressed in the circadian cells, as demonstrated by the accumulation of precursors of this miRNA when Dicer-1, drosha, or pasha was down-regulated in fly circadian tissues. Third, the effect of bantam (lengthening of the circadian period) resembles the period effect observed in flies carrying fewer genomic copies of clk, and it is opposite to the period effect observed in flies with additional clk copies. Fourth, the three evolutionarily conserved bantam sites are necessary for circadian rhythmicity. Nonetheless, the period effect due to bantam overexpression may be due to effects on other mRNAs in addition to clk (Kadener, 2009).
It is concluded that miRNAs have a role in the central pacemaker and, more specifically, that bantam regulates the central clock component clk. Whereas previous studies have identified miRNAs relevant to circadian rhythms, this one identifies a mRNA-miRNA pair involved in the core timekeeping process. Given the in vivo methods used to study miRNA function (including principally in neuronal tissue), it is suspected that they will have a broad impact on the study of miRNAs and their roles in regulating diverse aspects of Drosophila behavior (Kadener, 2009).
Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. This study presents evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and the Actin regulator Enabled was identified as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary (Becam, 2011).
Cell divisions lead to cell rearrangements that may challenge straight and sharp compartment boundaries. The DV boundary of mid- and late third instar wing primordia is characterized by a reduced rate of cell proliferation which defines the zone of non-proliferating cells (ZNC). The contribution of the ZNC to the maintenance of the DV affinity boundary was proposed many years ago but this notion was subsequently questioned. This study provides evidence that the ZNC does indeed play a role in boundary formation. bantam miRNA positively modulates the activity of the E2F transcription factor and drives G1-S transition in Drosophila tissues. Notch-mediated downregulation of bantam miRNA defines the ZNC and contributes to maintain a stable DV affinity boundary. Induction of proliferation in boundary cells by the ectopic expression of bantam, the cell cycle regulators Cyclin E and String, or the proto-oncogene dMyc, which is known to drive G1-S transition, compromises the formation of a smooth DV affinity boundary. A similar reduction in proliferation rates is observed at the rhombomere boundaries in the developing hindbrain, suggesting that reduced rate of cell proliferation might often be used in compartment boundary formation (Becam, 2011).
Notch-mediated downregulation of bantam activity is not only required to define the ZNC but also to establish the actomyosin cables observed at the interface between boundary and non-boundary cells. Ena, a regulator of Actin elongation, was identified as a direct target of bantam that is involved in DV boundary formation. The multiple roles of bantam in promoting G1-S transition and tissue growth, blocking apoptosis and regulating Actin dynamics unveil a new molecular connection between these three processes that might have relevance in growth control and tumorigenesis (Becam, 2011).
Intriguingly, bantam miRNA has no major role in the maintenance of the anterior-posterior compartment boundary of the developing wing and this boundary is not affected upon depletion of Ena protein levels. Thus, different regulators of actin elongation might be at work to regulate the actomyosin cytoskeleton and direct cell sorting in diverse developmental contexts. Whether reduced levels of bantam miRNA and increased levels of Ena protein are required to maintain differential cell sorting in the embryonic ectoderm or other imaginal tissues remains to be elucidated (Becam, 2011).
Cut is a late target of Notch that is expressed in boundary cells and is required to induce a stable Notch signaling center. This study demonstrate that Cut activity has also a specific function in reducing Ena mRNA and protein levels in boundary cells. Although depletion of Cut compromises the formation of the actomyosin cables at the interface of boundary and non-boundary cells and the maintenance of a stable DV affinity boundary, cell lineage and clonal analysis of wild-type and cut mutant cells reveal that Cut plays a major role in sorting boundary from non-boundary cells. The finding that the Notch signaling pathway defines, through Cut, a distinct population of boundary cells at the DV interface reinforces the mechanistic similarities in the maintenance of compartment boundaries within the vertebrate hindbrain and the Drosophila wing. In both developmental contexts, Notch defines a distinct population of boundary cells and contributes to segregating boundary from non-boundary cells. Although Cut mediates the role of Notch in the Drosophila wing, the molecular effectors mediating the role of vertebrate Notch in boundary formation remain uncharacterized. The data indicate that the later subdivision into boundary and non-boundary cells contributes to the maintenance of a stable DV affinity barrier in the mature wing primordium (Becam, 2011).
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).
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