Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. This study used loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. Expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process (Tan, 2011).

The patterns of developmental cell death are extremely complex and dynamic, and the fate of an individual cell probably represents the integration of multiple signaling pathways. Intrinsic pathways that define the identity and health of a cell, as well as extrinsic growth and survival pathways, are all likely to contribute to the life or death decision. In Drosophila, the transcriptional regulation of the RHG genes is an important output of the pathways that regulate cell death. The clustering of these genes to a single large genomic locus suggests that cell death signals can be integrated by regulatory sequences that control the coordinated expression of these genes (Tan, 2011).

This work shows that rpr, grim and skl are co-regulated to eliminate NBs during embryonic development. A genomic region important for this regulation was identified, located between rpr and grim. Identified genes or transcripts are surprisingly lacking in the 93 kb region between these genes, which is highly conserved between Drosophila species. This suggests that crucial cis-regulatory elements might be localized to this region (Tan, 2011).

The genomic region important for NB apoptosis was narrowed down to 22 kb. The number of NBs expressing grim and skl is significantly decreased in stage 14/15 embryos homozygous for a deletion of this region. The number of NBs expressing rpr is also affected, but to a smaller extent. Interestingly, there is still expression of grim, rpr and skl in NBs from stage 10 through stage 16. However, in the wild type, the number of NBs expressing all three genes increases at stage 14, presaging the loss of these cells by several hours. This increase in NBs expressing rpr, grim and skl is not seen in MM3 homozygous embryos. As overall grim, rpr and skl expression levels are not significantly altered in the mutants, the deleted regulatory sequences must control grim, rpr and skl expression in both a temporal- and tissue-specific manner, and baseline NB expression must be regulated by sequences outside of this region (Tan, 2011).

It is also interesting to note that the MM3 deletion has a more significant effect on grim and skl expression than on rpr expression. grim and skl are co-expressed in most cells of the stage 15 embryonic CNS, whereas rpr expression is less overlapping. As grim and skl lie 134 kb apart, on either side of rpr, long range enhancer interactions might regulate subsets of genes in this cell death locus. This type of long-range interaction has been implicated previously in the response to irradiation. Here again, a single regulatory region regulates multiple cell death genes at a great distance. The radiation responsive element activates hid and rpr, which are 200 kb apart, without having a major effect on the intervening gene grim. It will be important to examine higher order chromatin interactions to understand these long-range enhancer interactions (Tan, 2011).

The structure of the RHG cell death locus is conserved in other Drosophila species, but not obviously in more distantly related insect species, even though IAP inhibitory proteins are found in other species. This suggests that a coordinately regulated RHG gene cluster might be particularly important for Drosophila development (Tan, 2011).

As demonstrated by these studies, the individual roles for the cell death genes in developmental apoptosis are not well understood. Strong genetic and molecular data support a role for hid in regulating cell death in the embryonic head and in the developing eye. A role for grim in the death of microchaete glial cells has been described recently (Wu, 2010). Shown here is a specific role for grim in the elimination of the normal abdominal neuroblasts in the third instar larvae. As described in this work, rpr alone is not required for abdominal NB death in the embryo. However, this double mutant analysis demonstrates that rpr and grim must act together to eliminate these cells (Tan, 2011).

A possible role for hid in NB death cannot be eliminated, although no hid mRNA is detected in the VNC outside of the midline. A recent study suggests that rpr requires hid for efficient induction of apoptosis (Sandu, 2010). However, loss of hid does not result in increased NB survival, as would be expected if hid is required for rpr activity. Furthermore, loss of one copy of hid along with rpr and grim does not increase NB survival. Knockdown of rpr, grim and hid in NBs with an RHG miRNA also does not increase the number of surviving NBs over loss of rpr and grim. Further studies are needed to examine how hid and grim, rpr and skl differentially contribute to developmental apoptosis (Tan, 2011).

Although skl has similar pro-apoptotic activity to hid, grim and rpr, a role for skl in developmental apoptosis has not previously been demonstrated, owing to the lack of a skl mutation. This study shows that skl deletion alone does not alter NB death. In addition, it was found that deletion of one copy of skl along with grim and rpr slightly increase NB survival over deletion of grim and rpr alone. Deletion of skl and rpr, along with the NBRR in XR38/X20 also results in a larger VNC than deletion of rpr and the NBRR in XR38/H88, again suggesting a role for skl in cell death in this tissue. Additional double mutant analysis will be needed to test whether deletion of skl along with grim or rpr supports a role for skl in developmental apoptosis. Given the strongly overlapping expression of grim and skl in the embryonic VNC, it might be found that skl is redundant with grim in the CNS (Tan, 2011).

What are the developmental advantages of multiple cell death regulators? This work demonstrates that multiple RHG genes are activated within a single NB to activate apoptosis. In the salivary gland, hid and rpr, but not grim, are transcribed in the dying tissue, whereas the radiation response appears to involve hid, rpr and skl. These data suggest that the complex upstream regulation of cell death is likely to impact on different subsets of the RHG genes. If each gene is controlled by different combinations of enhancers this could provide greater flexibility in controlling the activation of cell death. In addition, a requirement for multiple cell death activators could ensure that cells are not killed inappropriately by the accidental activation of a single RHG gene (Tan, 2011).

The requirement for multiple RHG genes to kill cells might also reflect differences in the pro-apoptotic activities of these genes. For example, the RHG proteins show differential binding preferences to specific domains of the DIAP1 protein, and inhibit DIAP1 anti-apoptotic functions through different mechanisms. Additional activities, such as mitochondrial permeabilization, translational repression or interaction with other proteins such as Bruce or Scythe, also differ between RHG genes. Furthermore, post-translational regulation of particular RHG proteins might regulate their activity. This has been demonstrated for Hid; phosphorylation by MAPK downregulates the killing activity of Hid. It remains to be demonstrated whether different cell types have differential sensitivities to particular combinations of cell death genes when expressed at normal levels. This might provide yet another layer of control of the cell death process (Tan, 2011).

In sum, this work demonstrates that the regulation of NB apoptosis during development involves the coordinated temporal and spatial regulation of multiple cell death genes, controlled by a common regulatory region. By dissecting the RHG gene locus, specific genomic elements that regulate these genes in other developmentally important cell deaths are likely to be identified (Tan, 2011).

Characterization of the death-promoting activity of Sickle

A novel grim-reaper gene, termed sickle, has been identified that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression is detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression is detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death is antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression does not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an reaper/grim chimera or reaper (Wing, 2002).

To test sickle's ability to induce cell death, transient transfection assays were performed using cultured Spodoptera Sf-9 cells. Survival of the transfected cells was monitored using a LacZ reporter construct. Compared to the empty vector, transfection with the sickle expression construct results in a dramatic increase in cell death levels, as evidenced by an 18-fold reduction in LacZ expression. Transfection of a reaper expression construct also results in significant cell loss, although not to the same extent as is observed with sickle. The cell death induced by either sickle or reaper is suppressed ~3- to 6-fold by coexpression of the genes encoding the caspase inhibitory proteins p35 or DIAP1. These data imply that like other Grim-Reaper proteins, Sickle acts upstream of caspases and induces apoptosis via a mechanism involving inhibition of IAP function. The cell death-inducing capabilities of sickle were also investigated in Drosophila. Surprisingly, P[GMR-gal4]-targeted sickle expression using P[UAS-sickle] strains is ineffective at inducing ectopic cell death in the adult eye. Thus, P[GMR-gal4]/P[UAS-sickle] animals survive to adulthood, and nearly all exhibit normal eye morphology. (A few of these flies did have slightly roughened eyes, suggesting weak cell killing effects of sickle expression.) This result is in stark contrast to the lethality and complete loss of eye tissue seen for P[GMR-gal4]-targeted expression of reaper, hid, or grim. The use of several additional P[gal4] strains also failed to yield any evidence for sickle-induced ectopic cell death. While the basis for the distinct effects of sickle expression in cultured cells and Drosophila tissue is not yet clear, cell-specific effects of ectopic grim-reaper expression have been previously noted (Wing, 2002).

Because of the synergistic activities of reaper, hid, and grim in embryonic CNS midline, attempts were made to determine if sickle might enhance the actions of other grim-reaper genes. Since P[GMR-gal4]-targeted sickle expression failed to induce ectopic cell death, this issue was addressed by coexpression of either sickle and an reaper/grim chimera or reaper in the adult eye. P[GMR-gal4]-targeted expression of reaper/grim results in viable adults that exhibit a temperature-sensitive loss of eye tissue and pigmentation. In contrast, at either 25°C or 21°C P[GMR-gal4]-targeted coexpression of sickle and reaper/grim results in complete lethality. At 18°C, where the reaper/grim effects are reduced, a few flies coexpressing sickle and reaper/grim did emerge, and these exhibited a much greater loss of eye tissue than flies expressing either sickle or reaper/grim alone. Thus, sickle exhibits strong synergistic actions with reaper/grim. As expected, the effects of reaper/grim, as well as reaper/grim and sickle, are repressed by coexpression of p35; these animals are viable when raised at 25°C and exhibit essentially normal eye size and pigmentation. To demonstrate that sickle-dependent synergism is not restricted to the reaper/grim chimera, whether P[GMR-gal4]-targeted sickle expression would enhance cell death induced by P[GMR-reaper] was also examined. Flies bearing a single copy of P[GMR-reaper] exhibit a moderate loss of eye tissue. In contrast, flies bearing one copy each of P[GMR-reaper], P[GMR-gal4], and P[UAS-sickle], exhibit much more severe eye cell death, with greatly reduced eye size and pigmentation. This ectopic cell death is repressed by expression of p35. Synergistic eye cell death effects are also observed for coexpression of sickle and a g/reaper chimera, as well as sickle and grim. These results indicate that sickle can potentiate the cell killing effects of grim-reaper genes, and they provide the first examples of synergistic action for grim-reaper genes outside of the embryonic CNS midline (Wing, 2002).

In summary, these data demonstrate that sickle is a novel member of the grim-reaper family of cell death activators and suggest that functional interactions may be a general mechanism underlying the actions of Grim-Reaper proteins. The sequence of the Sickle protein strongly suggests that it has unique RHG motif-dependent and RHG motif-independent functions. Overall, the identification of sickle reveals further complexity in the regulation of cell death activation in Drosophila and provides additional evidence that these linked genes at 75C be considered a genetic complex (Wing, 2002).

The H99 chromosomal deficiency removes rpr, grim, and hid and prevents apoptotic cell death in embryos. Since skl resides close to a breakpoint mapped for this deletion, tests were performed to see whether expression of this locus might be affected in H99 homozygotes. H99 embryos are evidently normal for expression of skl, since a prominent signal for skl RNA, similar to the wild-type pattern, can be found in the CNS of H99 mutant embryos. Thus, unlike previously reported apoptogenic RNAs transcribed from within or outside of the Reaper region, skl expression is unperturbed in H99 embryos. As expected for animals that are completely cell death defective, wandering H99 macrophages show no positive signal for skl. Moreover, in stage 14/15 ventral nerve cords of H99 animals, up to four extra skl-expressing cells per hemisegment are detected. Thus, skl is expressed in at least some of the same cells that are fated to die through the action of rpr, grim, and hid (Christich, 2002).

To test the expression of skl in a model of precocious cell death, crumbs (crb) mutants were examined. In these embryos, widespread induction of rpr followed by ectopic apoptosis occurs throughout the epidermis. The signal for skl mRNA is detected in ectopically positioned epithelial cells, but the number and extent of positive cells is similar to wild-type embryos. This pattern is consistent with abnormal positioning of skl-positive cells and starkly contrasts with the widespread induction of rpr RNA seen in crb. Thus, unlike rpr, skl is not responsive to signals associated with premature apoptosis caused by this mutation (Christich, 2002).

Several lines of evidence suggest that skl is likely to be an enhancer rather than a determinant of PCD. (1) skl RNA does not completely coincide with all cells destined to die (expression is limited to only some zones of PCD in the embryo, and skl is not responsive when precocious cell death is prompted by distorted signaling). (2) Normal skl expression in H99 embryos excludes the possibility that lesions in skl might contribute to the global PCD defect found in these mutants. Therefore, endogenous skl expression alone is insufficient for embryonic apoptosis, since H99 animals are completely cell death defective. Consequently, normal skl expression alone is unlikely to specify cell death. However, tests were performed to see whether skl might provoke apoptosis when overexpressed in cultured SL2 cells; in this context, conditional expression of a full-length Skl cDNA triggers apoptosis, albeit with slightly less efficacy than Rpr. Unlike the N termini of Grim or Rpr, which are sufficient to provoke apoptosis when fused to GFP, the N terminus of Skl was negative for cell death in parallel assays. Thus, association between RHG proteins and IAPs is not necessarily sufficient to elicit apoptosis. Taken together, these observations suggest that skl might cooperate with other genes in the Reaper region to specify PCD in the embryo and could exert similar functions in developmental stages and tissues that were not examined (Christich, 2002).

Can the RHG motif of Skl bind to the BIR1 and BIR2 of DIAP1? Since the binding profile for the RHG motif of Rpr has not been reported, a Rpr(1-15)-GFP fusion protein was tested. In these studies, the first 15 residues of Skl and Rpr were expressed as N-terminal GFP fusion proteins in cultured fly cells. Lysates containing Skl(1-15)-GFP or Rpr(1-15)-GFP were incubated with recombinant GST-BIR1 or GST-BIR2 on glutathione beads. Skl(1-15)-GFP specifically binds to the BIR2 domain DIAP1. A similar binding profile is observed with Rpr(1-15)-GFP lysates. Skl-derived or Rpr-derived residues are required for this association, since GFP alone does not bind. Binding to the BIR1 domain by either of these fusion proteins was not detected. These results parallel binding reported for N-terminal peptides of Hid and Grim, which form stable complexes with the BIR2 domain but not the BIR1 domain of DIAP1. Since it is induced by ionizing radiation, skl might also exert functions as part of a genotoxic stress response. For instance, skl might cooperate with rpr to promote apoptosis in response to damage signals, or it could act as a sentinel protein to increase sensitization to cellular damage, as recently suggested for Omi/HtrA2. This possibility was tested by assessing the effects of either full-length Skl or Skl(1-15)-GFP in irradiated SL2 cells, but preliminary results from these analyses were negative. Thus, if Skl is a sensitizing protein, it may function in a cell type-specific manner (Christich, 2002).

Principles of microRNA-target recognition: Targeting of sickle 3'UTR by microRNAs

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. This study systematically evaluates the minimal requirements for functional miRNA-target duplexes in vivo and classes of target sites with different functional properties are distinguished. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. Examples and genome-wide statistical support is presented to show that both classes of sites are used in biologically relevant genes. Evidence is provided that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families (Brennecke, 2005).

To improve understanding of the minimal requirements for a functional miRNA target site, use was made of a simple in vivo assay in the Drosophila wing imaginal disc. A miRNA was expressed in a stripe of cells in the central region of the disc and its ability to repress the expression of a ubiquitously transcribed enhanced green fluorescent protein (EGFP) transgene containing a single target site in its 3' UTR was assessed. The degree of repression was evaluated by comparing EGFP levels in miRNA-expressing and adjacent non-expressing cells. Expression of the miRNA strongly reduced EGFP expression from transgenes containing a single functional target site (Brennecke, 2005).

In a first series of experiments it was asked which part of the RNA duplex is most important for target regulation. A set of transgenic flies was prepared, each of which contained a different target site for miR-7 in the 3' UTR of the EGFP reporter construct. The starting site resembled the strongest bantam miRNA site in its biological target hid and conferred strong regulation when present in a single copy in the 3' UTR of the reporter gene. The effects were tested of introducing single nucleotide changes in the target site to produce mismatches at different positions in the duplex with the miRNA (note that the target site mismatches were the only variable in these experiments). The efficient repression mediated by the starting site was not affected by a mismatch at positions 1, 9, or 10, but any mismatch in positions 2 to 8 strongly reduced the magnitude of target regulation. Two simultaneous mismatches introduced into the 3' region had only a small effect on target repression, increasing reporter activity from 10% to 30%. To exclude the possibility that these findings were specific for the tested miRNA sequence or duplex structure, the experiment was repeated with miR-278 and a different duplex structure. The results were similar, except that pairing of position 8 was not important for regulation in this case. Moreover, some of the mismatches in positions 2-7 still allowed repression of EGFP expression up to 50%. Taken together, these observations support previous suggestions that extensive base-pairing to the 5' end of the miRNA is important for target site function (Brennecke, 2005).

Next the minimal 5' sequence complementarity necessary to confer target regulation was determined. The core of 5' sequence complementarity essential for target site recognition is referred to as the 'seed'. All possible 6mer, 5mer, and 4mer seeds complementary to the first eight nucleotides of the miRNA were tested in the context of a site that allowed strong base-pairing to the 3' end of the miRNA. The seed was separated from a region of complete 3' end pairing by a constant central bulge. 5mer and 6mer seeds beginning at positions 1 or 2 are functional. Surprisingly, as few as four base-pairs in positions 2-5 confers efficient target regulation under these conditions, whereas bases 1-4 are completely ineffective. 4mer, 5mer, or 6mer seeds beginning at position 3 are less effective. These results suggest that a functional seed requires a continuous helix of at least 4 or 5 nucleotides and that there is some position dependence to the pairing, since sites that produce comparable pairing energies differ in their ability to function. These experiments also indicate that extensive 3' pairing of up to 17 nucleotides in the absence of the minimal 5' element is not sufficient to confer regulation. Consequently, target searches based primarily on optimizing the extent of base-pairing or the total, and ranking miRNA target sites according to overall complementarity or free energy of duplex formation might not reflect their biological activity (Brennecke, 2005).

To determine the minimal lengths of 5' seed matches that are sufficient to confer regulation alone, single sites were tested that pair with eight, seven, or six consecutive bases to the miRNA's 5' end, but that do not pair to its 3' end. Surprisingly, a single 8mer seed (miRNA positions 1-8) is sufficient to confer strong regulation by the miRNA. A single 7mer seed (positions 2-8) is also functional, although less effective. The magnitude of regulation for 8mer and 7mer seeds is strongly increased when two copies of the site are introduced in the UTR. In contrast, 6mer seeds show no regulation, even when present in two copies. Comparable results have been reported for two copies of an 8mer site with limited 3' pairing capacity in a cell-based assay. These results do not support a requirement for a central bulge (Brennecke, 2005).

From these experiments it is concluded that (1) complementarity of seven or more bases to the 5' end miRNA is sufficient to confer regulation, even if the target 3' UTR contains only a single site; (2) sites with weaker 5' complementarity require compensatory pairing to the 3' end of the miRNA in order to confer regulation, and (3) extensive pairing to the 3' end of the miRNA is not sufficient to confer regulation on its own without a minimal element of 5' complementarity (Brennecke, 2005).

While recognizing that there is a continuum of base-pairing quality between miRNAs and target sites, the experiments presented here suggest that sites that depend critically on pairing to the miRNA 5' end (5' dominant sites) can be distinguished from those that cannot function without strong pairing to the miRNA 3' end (3' compensatory sites). The 3' compensatory group includes seed matches of four to six base-pairs and seeds of seven or eight bases that contain G:U base-pairs, single nucleotide bulges, or mismatches (Brennecke, 2005).

It is useful to distinguish two subgroups of 5' dominant sites: those with good pairing to both 5' and 3' ends of the miRNA (canonical sites) and those with good 5' pairing but with little or no 3' pairing (seed sites). Seed sites are considered to be those where there is no evidence for pairing of the miRNA 3' end to nearby sequences that is better than would be expected at random. The possibility cannot be excluded that some sites identified as seed sites might be supported by additional long-range 3' pairing. Computationally, this is always possible if long enough loops in the UTR sequence are allowed. Whether long loops are functional in vivo remains to be determined (Brennecke, 2005).

Canonical sites have strong seed matches supported by strong base-pairing to the 3' end of the miRNA. Canonical sites can thus be seen as an extension of the seed type (with enhanced 3' pairing in addition to a sufficient 5' seed) or as an extension of the 3' compensatory type (with improved 5' seed quality in addition to sufficient 3' pairing). Individually, canonical sites are likely to be more effective than other site types because of their higher pairing energy, and may function in one copy. Due to their lower pairing energies, seed sites are expected to be more effective when present in more than one copy (Brennecke, 2005).

Most currently identified miRNA target sites are canonical. For example, the hairy 3' UTR contains a single site for miR-7, with a 9mer seed and a stretch of 3' complementarity. This site has been shown to be functional in vivo , and it is strikingly conserved in the seed match and in the extent of complementarity to the 3' end of miR-7 in all six orthologous 3' UTRs (Brennecke, 2005).

Although seed sites have not been previously identified as functional miRNA target sites, there is some evidence that they exist in vivo. For example, the Bearded (Brd) 3' UTR contains three sequence elements, known as Brd boxes, that are complementary to the 5' region of miR-4 and miR-79. Brd boxes have been shown to repress expression of a reporter gene in vivo, presumably via miRNAs; expression of a Brd 3' UTR reporter is elevated in dicer-1 mutant cells, which are unable to produce any miRNAs. All three Brd box target sites consist of 7mer seeds with little or no base-pairing to the 3' end of either miR-4 or miR-79. The alignment of Brd 3' UTRs shows that there is little conservation in the miR-4 or miR-79 target sites outside the seed sequence, nor is there conservation of pairing to either miRNA 3' end. This suggests that the sequences that could pair to the 3' end of the miRNAs are not important for regulation as they do not appear to be under selective pressure. This makes it unlikely that a yet unidentified Brd box miRNA could form a canonical site complex (Brennecke, 2005).

The 3' UTR of the HOX gene Sex combs reduced (Scr) provides a good example of a 3' compensatory site. Scr contains a single site for miR-10 with a 5mer seed and a continuous 11-base-pair complementarity to the miRNA 3' end. The miR-10 transcript is encoded within the same HOX cluster downstream of Scr, a situation that resembles the relationship between miR-iab-5p and Ultrabithorax in flies and miR-196/HoxB8 in mice. The predicted pairing between miR-10 and Scr is perfectly conserved in all six drosophilid genomes, with the only sequence differences occurring in the unpaired loop region. The site is also conserved in the 3' UTR of the Scr genes in the mosquito, Anopheles gambiae, the flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori. Conservation of such a high degree of 3' complementarity over hundreds of millions of years of evolution suggests that this is likely to be a functional miR-10 target site. Extensive 5' and 3' sequence conservation is also seen for other 3' compensatory sites, e.g., the two let-7 sites in lin-41 or the miR-2 sites in grim and sickle (Brennecke, 2005).

Several families of miRNAs have been identified whose members have common 5' sequences but differ in their 3' ends. In view of the evidence that 5' ends of miRNA are functionally important, and in some cases sufficient, it can be expected that members of miRNA families may have redundant or partially redundant functions. According to this model, 5' dominant canonical and seed sites should respond to all members of a given miRNA family, whereas 3' compensatory sites should differ in their sensitivity to different miRNA family members depending on the degree of 3' complementarity. This is being tested using the wing disc assay with 3' UTR reporter transgenes and overexpression constructs for various miRNA family members (Brennecke, 2005).

miR-4 and miR-79 share a common 5' sequence that is complementary to a single 8mer seed site in the bagpipe 3' UTR. The 3' ends of the miRNAs differ. miR-4 is predicted to have 3' pairing at approximately 50% of the maximally possible level (~10.8 kcal/mol), whereas the level of 3' pairing for miR-79 is approximately 25% maximum (~6.1 kcal/mol), which is below the average level expected for random matches. Both miRNAs repressed expression of the bagpipe 3' UTR reporter, regardless of the 3' complementarity. This indicates that both types of site are functional in vivo and suggests that bagpipe is a target for both miRNAs in this family (Brennecke, 2005).

To test whether miRNA family members can also have non-overlapping targets, 3' UTR reporters were used of the pro-apoptotic genes grim and sickle, two recently identified miRNA targets. Both genes contain K boxes in their 3' UTRs that are complementary to the 5' ends of the miR-2, miR-6, and miR-11 miRNA family. These miRNAs share residues 2-8 but differ considerably in their 3' regions. The site in the grim 3' UTR is predicted to form a 6mer seed match with all three miRNAs, but only miR-2 shows the extensive 3' complementarity that would be needed for a 3' compensatory site with a 6mer seed to function (~19.1 kcal/mol, 63% maximum 3' pairing, versus ~10.9 kcal/mol, 46% maximum, for miR-11 and ~8.7 kcal/mol, 37% maximum, for miR-6). Indeed, only miR-2 is able to regulate the grim 3' UTR reporter, whereas miR-6 and miR-11 are non-functional (Brennecke, 2005).

The sickle 3' UTR contains two K boxes and provides an opportunity to test whether weak sites can function synergistically. The first site is similar to the grim 3' UTR in that it contains a 6mer seed for all three miRNAs but extensive 3' complementarity only to miR-2. The second site contains a 7mer seed for miR-2 and miR-6 but only a 6mer seed for miR-11. miR-2 strongly downregulates the sickle reporter, miR-6 has moderate activity (presumably via the 7mer seed site), and miR-11 has nearly no activity, even though the miRNAs were overexpressed. The fact that a site is targeted by at least one miRNA argues that it is accessible (e.g., miR-2 is able to regulate both UTR reporters), and that the absence of regulation for other family members is due to the duplex structure. These results are in line with what would be expected based on the predicted functionality of the individual sites, and indicate that the model of target site functionality can be extended to UTRs with multiple sites. Weak sites that do not function alone also do not function when they are combined (Brennecke, 2005).

To show that endogenous miRNA levels regulate all three 3' UTR reporters, EGFP expression was compared in wild-type cells and dicer-1 mutant cells, which are unable to produce miRNAs. dicer-1 clones did not affect a control reporter lacking miRNA binding sites, but showed elevated expression of a reporter containing the 3' UTR of the previously identified bantam miRNA target hid. Similarly, all 3' UTR reporters above were upregulated in dicer-1 mutant cells, indicating that bagpipe, sickle, and grim are subject to repression by miRNAs expressed in the wing disc. Taken together, these experiments indicate that transcripts with 5' dominant canonical and seed sites are likely to be regulated by all members of a miRNA family. However, transcripts with 3' compensatory sites can discriminate between miRNA family members (Brennecke, 2005).

Experimental tests such as those presented in this study and the observed evolutionary conservation suggest that all three types of target sites are likely to be used in vivo. To gain additional evidence the occurrence of each site type was examined in all Drosophila 3' UTRs. Use was made of the D. pseudoobscura genome, the second assembled drosophilid genome, to determine the degree of site conservation for the three different site classes in an alignment of orthologous 3' UTRs. From the 78 known Drosophila miRNAs, a set of 49 miRNAs with non-redundant 5' sequences was chosen. Whether sequences complementary to the miRNA 5' ends are better conserved than would be expected for random sequences was tested. For each miRNA, a cohort of ten randomly shuffled variants was constructed. To avoid a bias for the number of possible target matches, the shuffled variants were required to produce a number of sequence matches comparable (±15%) to the original miRNAs for D. melanogaster 3' UTRs. 7mer and 8mer seeds complementary to real miRNA 5' ends were significantly better conserved than those complementary to the shuffled variants. Conserved 8mer seeds for real miRNAs occur on average 2.8 times as often as seeds complementary to the shuffled miRNAs. For 7mer seeds this signal was 2:1, whereas 6mer, 5mer, and 4mer seeds did not show better conservation than expected for random sequences. To assess the validity of these signals and to control for the random shuffling of miRNAs, this procedure was repeated with 'mutant' miRNAs in which two residues in the 5' region were changed. There was no difference between the mutant test miRNAs and their shuffled variants. This indicates that a substantial fraction of the conserved 7mer and 8mer seeds complementary to real miRNAs identify biologically relevant target sites (Brennecke, 2005).

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development: Differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle

MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).

miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).

One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).

To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).

The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).

The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).

To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).

To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).

To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).

The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).

Protein Interactions

To examine the interactions between DIAP1 and Skl, the second BIR domain (BIR2) was crystallized in complex with an N-terminal 10 residue peptide of Skl. The structure was determined at 2.1 Å resolution. The final atomic model of DIAP1-BIR2 contains residues 215–316, comprising six alpha helices, a three-stranded ß sheet, and a zinc atom chelated by 3 Cys and 1 His residues (Cys263, Cys266, Cys290, and His283). This structure is nearly identical to that complexed with Hid or Grim peptide, with root-mean-square deviation (RMSD) of less than 0.2 Å for all aligned Calpha atoms. Binding by the Skl peptide, however, differs from that of a Hid or Grim peptide. In contrast to the conserved DIAP1 binding by 7 residues in the Grim or Hid peptide, only 4 N-terminal residues of the Skl peptide bind a shallow surface groove on DIAP1, consistent with limited sequence homology between Skl and the other three Drosophila death proteins (Srinivasula, 2002).

Binding to the DIAP1-BIR2 domain results in the burial of 600 Å2 of surface area for the Skl peptide. The recognition involves both hydrogen bond networks and van der Waals contacts between 4 hydrophobic residues on the peptide and conserved DIAP1 residues. Similar to the Smac/DIABLO-XIAP interactions, the N terminus of Skl is positioned in an acidic environment, in which 3 charged residues (Asp277, Gln282, and Glu314) in DIAP1 play an essential role in binding the peptide. The amino group of Ala1 donates two hydrogen bonds to the surrounding residues Asp277 and Gln282, while the carbonyl group accepts two hydrogen bonds from the side chains of Trp286 and Glu314. These central interactions are buttressed by additional hydrogen bonds and hydrophobic contacts (Srinivasula, 2002).

Whether the RHG motif of Skl could bind to the BIR1 and BIR2 of DIAP1 was tested. Since the binding profile for the RHG motif of Rpr has not been reported, a Rpr(1-15)-GFP fusion protein was tested. In these studies, the first 15 residues of Skl and Rpr were expressed as N-terminal GFP fusion proteins in cultured fly cells. Lysates containing Skl(1-15)-GFP or Rpr(1-15)-GFP were incubated with recombinant GST-BIR1 or GST-BIR2 on glutathione beads. Skl(1-15)-GFP specifically binds to the BIR2 domain DIAP1. A similar binding profile is observed with Rpr(1-15)-GFP lysates. Skl-derived or Rpr-derived residues are required for this association, since GFP alone does not bind. Binding to the BIR1 domain by either of these fusion proteins was not detected. These results parallel binding reported for N-terminal peptides of Hid and Grim, which form stable complexes with the BIR2 domain but not the BIR1 domain of DIAP1 (Christich, 2002).



sickle transcripts ar detected beginning at stage 11 of embryogenesis in symmetric dorsal anterior regions in the prospective head region. Slightly later, sickle mRNA is also detected in ventral anterior regions of the head, as well as in cells of the neurogenic ectoderm. sickle continues to be expressed in cells within the developing brain and ventral nerve cord during later stages of embryogenesis. Overall, this pattern is similar to that of dying cells within the nervous system, and sickle mRNA is also detected within phagocytic macrophages. sickle gene expression was further analyzed via reverse transcriptase/polymerase chain reaction assays, which indicated that sickle is expressed in third instar larvae, pupae, and adult heads. Expression of sickle was also investigated in Df(3L)H99 mutants. Df(3L)H99 is the smallest known deletion that removes reaper, hid, and grim, and the right breakpoint maps close to the sickle gene. PCR assays initially indicate that DNA sequences corresponding to the coding region of sickle but not reaper are still present in homozygous Df(3L)H99 mutants. Whole mount in situ hybridizations demonstrate that the sickle gene is expressed in Df(3L)H99 mutant embryos. Significantly, much more widespread expression of sickle mRNA is detected in the nervous system of Df(3L)H99 mutants than in wild-type embryos. This ectopic sickle expression is likely the result of an accumulation of sickle mRNA in 'rescued' neural cells that fail to die in Df(3L)H99 mutants and are not removed via phagocytosis. These data indicate that in the absence of other grim-reaper genes, sickle is largely insufficient to permit embryonic cell deaths to occur. However, the data do not preclude an essential role for sickle in cell death activation. Thus, sickle could be crucial for the cell deaths that still take place in Df(3L)H99 mutants, or sickle could function predominantly in concert with reaper, hid, or grim. Importantly, as mutations disrupting hid or hid and grim yield much less severe cell death phenotypes than does Df(3L)H99, elucidation of sickle functions will require determination of the phenotypes of both sickle-specific mutants as well as mutants that eliminate sickle and other grim-reaper genes (Wing, 2002).

As a first step in determining the function of skl in Drosophila, the expression of skl mRNA was examined by RT-PCR analysis at different stages of Drosophila embryonic development. This analysis revealed that skl mRNA is developmentally regulated. It is initially present in small amounts in 4-14 hr embryos and in larger amounts in 14-21 hr embryos. No skl mRNA was detected in adult male or female flies. Interestingly this temporal expression profile is similar to that of Reaper, suggesting that the two genes are under similar developmental regulation. It is noteworthy that most developmentally programmed cell death in Drosophila occurs during this period of embryonic development, suggesting that Skl could play an important role in this process (Srinivasula, 2002).

The mRNA and protein expression patterns were examined in whole Drosophila embryos. Expression in embryos is localized to specific regions of the head and to specific cells of the central nervous system (CNS). The CNS expression pattern is highly reminiscent of those of the cell death inducers Reaper and Grim, while dorsal head staining may foreshadow that of Hid in the optic lobe region. The similarity of the Skl expression pattern to those of the known cell death inducers (Reaper and Grim) suggests that it may act in concert with these proteins to orchestrate the complex pattern of apoptosis during development, particularly in the CNS (Srinivasula, 2002).

In situ hybridization was used to characterize skl expression during embryogenesis. skl mRNA is closely associated with some but not all areas where programmed cell death (PCD) occurs. Earliest skl expression can be detected in germ band extended embryos (stage 10). At this time, expression is rather ubiquitous but is preferentially found in the epidermis of the embryo. In stages 11 through 13, skl transcripts accumulate in a distinctly punctate pattern in the dorsal aspect of the head. At this time, skl RNAs also occur in scattered patterns near the pharynx, the hindgut, and throughout the lateral epidermis. In stages 14 and beyond, expression is predominantly found within subsets of cells in the ventral nerve cord and the brain. At high magnification, skl transcripts are detected subcellularly localized inside some but not all macrophages. As has been demonstrated with probes for rpr, hid, grim, and dredd, this characteristic signal within macrophages reflects hybridization to mRNAs inside cell corpses that were recently engulfed by phagocytic cells. Together, these observations show that skl expression has an apoptogenic component that is clearly associated with PCD in the embryo. However, in contrast to rpr, grim, and hid, PCD-associated expression of skl is limited to only some rather than all zones of PCD. For instance, skl expression correlates well with PCD in the head region but is clearly absent from a prominent zone of apoptosis in the ventral epidermis of stage 12 to 13 embryos. skl might also exert functions in developmental processes unrelated to PCD (Christich, 2002).


Brennecke, J., Stark, A., Russell, R. B. and Cohen, S. M. (2005). Principles of microRNA-target recognition. PLoS Biol. 3(3):e85. 15723116

Christich, A., et al. (2002). The damage-responsive Drosophila gene sickle encodes a novel IAP reaper, grim, and hid Curr. Biol. 12: 137-140. 11818065

Leaman, D., et al. (2005). Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121: 1097-1108. 15989958

Sandu, C., Ryoo, H. D. and Steller, H. (2010). Drosophila IAP antagonists form multimeric complexes to promote cell death. J. Cell Biol. 190: 1039-1052. PubMed Citation: 20837774

Srinivasula, S. M., et al. (2002). sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein Curr. Biol. 12: 125-130. 11818063

Tan, Y., et al. (2011). Coordinated expression of cell death genes regulates neuroblast apoptosis. Development 138(11): 2197-206. PubMed Citation: 21558369

Wing, J. P., et al. (2002). Drosophila sickle is a novel grim-reaper cell death activator. Curr. Biol. 12: 131-135. 11818064

Wu, J. N., et al. (2010). grim promotes programmed cell death of Drosophila microchaete glial cells. Mech. Dev. 127: 407-417. PubMed Citation: 20558283

sickle: Biological Overview | Regulation | Developmental Biology

date revised: 1 June 2002

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.