An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).
The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).
rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).
During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).
The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).
To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).
Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notchts1 allele was used. However, when Notchts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notchts1/Notch55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).
Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).
In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).
In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. The Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. These results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons (Miguel-Aliaga, 2004).
How does Abd-B prevent the function of RHG-motif genes? It is likely that Abd-B prevents pioneer neuron apoptosis by repressing the transcription of, at least, rpr and grim. This idea is supported by four facts: (1) the H99 deletion is epistatic to (functions downstream of) Abd-B; (2) Abd-B is a transcription factor; (3) rprGAL4 is activated posteriorly in Abd-Bm mutants; (4) when misexpressed postmitotically, Abd-B can fully rescue both types of pioneer neurons. Given that loss of rpr is critical for anterior dMP2 survival, whereas loss of grim is critical for anterior MP1s, Abd-B must prevent the expression of at least these two cell death activators (Miguel-Aliaga, 2004).
The combined activity of RHG-motif genes is critical to the initiation of all cell death in the Drosophila embryo. These genes act in an additive manner. However, not all cell death activators are simultaneously expressed in every cell fated to die, and their specific expression patterns do not always overlap. Therefore, it is likely that they are differentially regulated by specific developmental signals. While Abd-B acts to repress rpr and grim function in posterior pioneer neurons, the developmental stimulus activating their expression in these neurons throughout the cord is currently unknown. Three developmental signals are known to regulate the function of RHG-motif genes in the Drosophila nervous system. The insect hormone ecdysone appears to be important for blocking cell death of certain peptidergic neurons during metamorphosis. However, the ecdysone-receptor complex has also been shown to promote cell death by activating rpr transcription in other tissues during Drosophila metamorphosis. While an embryonic ecdysone pulse occurs around the time when pioneer neurons die, preliminary experiments have failed to lend any support to an ecdysone-dependent activation of apoptosis in these neurons. The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate Hid protein, thereby preventing apoptosis of midline glial cells. However, neither Rpr nor Grim appear to be regulated in this fashion, and this model would not address the specific transcriptional activation of these genes in pioneer neurons. Lastly, Notch signaling has been described as resulting in both activation and inhibition of apoptosis. In Drosophila, recent studies have revealed that Notch can act cell-autonomously to induce apoptosis during final mitotic divisions both in the central and peripheral nervous systems. Although this Notch-induced developmental apoptosis is prevented in H99 mutant embryos, the molecular mechanisms by which activated Notch signaling results in the activation of IAP inhibitors are still unknown. Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since it is not active in dMP2 neurons. It is, therefore, likely that an as yet unidentified factor is responsible for the activation of the apoptotic machinery in pioneer neurons. This factor could be Odd-skipped, given its specific expression in dMP2 and MP1 neurons. Because of the early role of odd in embryonic patterning, its possible postmitotic function in these neurons cannot be addressed using the currently available odd mutants (Miguel-Aliaga, 2004).
Developmental apoptosis in invertebrate embryos typically occurs shortly after cells are generated. In Drosophila, this has often precluded the identification of dying cells until apoptosis has been genetically prevented. Consequently, progress in identification of the mechanisms controlling apoptosis has been relatively slow, and little is known about the upstream pathways that initiate cell death in specific tissues or lineages. Furthermore, in the Drosophila VNC, studies have shown that apoptotic corpses are engulfed by glia, transported to the dorsal surface of the VNC and transferred to macrophages for final destruction. The molecular genetic mechanisms underlying this intriguing series of events are only just beginning to be unraveled. The identification of a late apoptotic event in two of the best-studied and least complex lineages in the Drosophila CNS, as well as the characterization of the dMP2-GAL4 line, should contribute to the elucidation of the mechanisms involved in both the developmental initiation and execution of apoptosis (Miguel-Aliaga, 2004).
MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs. The genomes of metazoans encode on the order of several hundred miRNAs, but the processes they regulate have been defined for only a few cases. New inhibitors of apoptotic cell death were sought by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. The Drosophila miRNA mir-14 has been identified as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. mir-14 mutants have elevated levels of the apoptotic effector caspase Ice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect (Xu, 2003).
The two C. elegans miRNAs with known functions, lin-4 and let-7, are thought to regulate development by binding to the 3'untranslated region of target transcripts and thereby repressing the translation of their products. In these examples, the analysis of genetic interactions provides important clues as to the identity of targets. In the absence of this sort of information, it is difficult to predict miRNA targets in animals. This is because base pairing between the mature miRNA and its target is imperfect and the rules that govern which base pair interactions are important are unknown. Potential Mir-14 binding sites were sought in a number of apoptotic regulators, including Dronc, Rpr, Hid, and Grim. Potential target sites were identified in the transcripts of several genes, including Ice, Dcp-1, Scythe, SkpA, and Grim (however, the Grim target is present in the 3'UTR, which was absent in the GMR-Grim transgene). Of these, Ice, an apoptotic effector caspase, is of particular interest. Ice is required for at least some cell deaths and is activated by Dronc, which promotes cell death induced by Rpr, Hid, and Grim. Ice levels in adults were measured by using an anti-Ice antibody. Ice is elevated in mir-14Δ1 flies as compared to the wild-type, and this increase is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment. Whereas these observations alone do not prove that Ice is a direct target of Mir-14, they do suggest that Ice is regulated, either directly or indirectly, by Mir-14 levels (Xu, 2003).
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).
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).
Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. grim expression is shown to induce apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. The conserved N-terminal domain is not required for either the initiation or the execution of apoptosis by Grim. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. Grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Three clustered aspartate residues near the Grim C-terminus (positions 126, 128 and 129) could then be used by a caspase as a target sequence. The downshift predicted by digestion at these sites is between 1 and 1.3 kDa; however, the observed size for the short form is ~4 kDa less than that observed for the intact protein. Nevertheless, C-terminal deletions of the protein provoke downshifts greater than expected, which are in the range of the observed downshift for the proteolysed form of the protein. These results show that besides activating caspases, Grim itself can be processed as a consequence of caspase proteolytic activity. Even though inhibitors of apoptosis OpIAP and DEVD.cho do not block Grim-induced apoptosis, they are both functionally active in repressing endogenous caspases in either treated or transfected cells, since they prevented Grim cleavage. Therefore, it is likely that caspases insensitive to OpIAP and/or DEVD.cho are sufficient to achieve Grim-induced apoptosis (Clavería, 1998).
Grim-induced death is antagonized by bcl-2 in a dose-dependent manner; neither Fas signaling nor p53 are required for Grim pro-apoptotic activity. The sensitivity of the grim-induced death to bcl-2 levels suggests that Grim acts by activating a mitochondrial pathway. To determine the site of Grim action, its subcellular localization was explored in transfected cells prior to the time they show morphological symptoms of apoptosis. Pre-apoptotic fibroblasts simultaneously show Grim in two different cytoplasmic localizations: diffuse cytosolic and a punctate pattern. However, while Grim-transfected cells show an almost exclusive cytosolic localization, cells treated with cell-permeable broad specificity caspase inhibitor zVAD.fmk show predominantly the punctate pattern. The same pattern is observed in Grim-expressing p35-rescued cells. Co-localization of Grim protein with a mitochondrial-specific antibody shows that the punctate pattern corresponds to mitochondria. It is concluded that Grim localizes initially in the cytoplasm but accumulates progressively in the mitochondria, correlating with apoptosis progression. It is possible that Grim translocation to mitochondria is the event that triggers the apoptotic pathway. In that case, the increased mitochondrial localization of Grim in zVAD-treated cells would be the result of the apoptosis blockade by zVAD at a point downstream of Grim incorporation in the mitochondria. These results show that Drosophila Grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases (Clavería, 1998).
It is possible that mitochondrial components of the mammalian cellular apoptotic machinery recognize the Drosophila Grim protein and respond by activating the apoptotic programme, as they would after the proper endogenous stimulus. In good agreement with this view, Grim action is counteracted by the overexpression of the anti-apoptotic factor bcl-2, which generally inhibits all mitochondrial death pathways. bcl-2 inhibits Grim-induced death in a dose-dependent manner, suggesting that they mutually antagonize to either promote or inhibit caspase activation. A bcl-2-like molecule has not yet been isolated in Drosophila, but mammalian and nematode members of this family have been shown to rescue ced-3- and Reaper-induced apoptosis in cultured fly cells. One possibility is that Grim promotes cytochrome c release from the mitochondria, as has been shown for Reaper in an in vitro amphibian system. However, Reaper does not induce apoptosis in mouse fibroblasts, arguing for a mechanism of action or regulation different from that of Grim. The activity of caspases insensitive to OpIAP, DEVD.cho and YVAD.cho is sufficient to execute the apoptosis induced by Grim. Caspase 9, which has been shown to transduce apoptotic mitochondrial signals, may be one of those activated by mitochondrial Grim. Nevertheless, group II caspases are activated as well, since Grim cleavage is inhibited by caspase inhibitors specific for this group and it cannot be excluded that activation of group II caspases may also serve to execute Grim-induced apoptosis. Inhibitor of apoptosis proteins (IAPs), the natural inhibitors of group II caspases, have been shown to inhibit apoptosis by directly binding to Grim, Reaper and Hid in a lepidopteran cell line. In contrast, neither OpIAP nor DEVD block Grim-induced death. These results, nevertheless, show that group II caspases are activated, suggesting that some of the Grim-induced caspase activation cascades are similar between mouse fibroblasts and cultured lepidopteran cells, whereas others may differ. It is possible that the particular caspase activation cascade triggered by grim depends more on the cell type in which it is expressed rather than on any intrinsic specificity of its action (Claveria, 1998).
Mutations that remove Nedd2-like caspase (DRONC) are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).
Ectopic expression of Reaper or Grim induces substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis is inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrate that both Reaper and Grim physically interact with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolishes binding to cIAPs. In vitro binding experiments indicate that Reaper and Grim bind specifically to the BIR domain-containing region of cIAPs, since deletion of this region results in loss of binding. The physical interaction has been further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localize with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolishes co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, it has been demonstrated that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain, which is the only region having significant homology between these two Drosophila death inducers (McCarthy, 1998).
Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact with Rpr through IAP family members' BIR (baculovirus IAP repeat) motifs. Transient overexpression of HID and GRIM also induces apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs (see Drosophila Thread) block HID- and GRIM-induced apoptosis and also physically interact with them through the BIR motifs of the IAPs. The region of sequence similarity shared by Rpr, Hid, and Grim (the N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins (Vucic, 1998).
Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).
It is thought that the previously proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems. When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal mechanism of action of these proteins in the cell death pathway (Goyal, 2000).
The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).
Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases drICE and DCP-1 and to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).
These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th109.07, which lacks most of the protein, as well as for th5 and th4, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase drICE. The th9 mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).
It is interesting to note that th7, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).
Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The thSL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH2-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).
The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th6B and th81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).
In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).
In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).
Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).
Many members of the inhibitor of apoptosis (IAP) family of proteins suppress programmed cell death, at least in part, by physically interacting with and inhibiting the catalytic activity of caspases. An important functional unit in all death-inhibiting IAP proteins is the so-called baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. The Drosophila genome contains four genes that encode proteins with BIR domains. The overexpression of two of these, DIAP1 and DIAP2, inhibit both normal developmental cell death and apoptosis induced by expression of proapoptotic genes. In addition, DIAP1 is required for cell survival in the embryo and in a number of adult tissues. These observations, in conjunction with others showing that DIAP1 binds and inactivates several Drosophila caspases and that loss of DIAP1 results in an increase in caspase activity in vivo, argue that DIAP1's function as a caspase inhibitor is required for cell survival. DIAP1 contains two N-terminal BIR repeats and a C-terminal RING domain. DIAP1 fragments containing the BIR2 domain are sufficient to prevent cell death in a number of contexts. Interestingly, fragments consisting of the BIR2 and surrounding linker sequences also bind multiple proapoptotic proteins, including the apical caspase DRONC, and Hid, Grim, and Reaper (Wu, 2001 and references therein).
One mechanism by which Hid, Grim, and Reaper promote cell death is by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor. Although Hid, Grim, and Reaper perform a similar function in promoting cell death, they only share homology in the N-terminal 14 residues of their primary sequences. These N-terminal sequences are sufficient to mediate interactions with DIAP1 and with several mammalian IAPs. In the case of Hid in insects, and Hid and Reaper in mammalian cells, these N-terminal sequences are essential for proapoptotic function (Wu, 2001 and references therein).
In mammalian cells, caspase inhibition by IAPs is negatively regulated by a mitochondrial protein Smac/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol upon apoptotic stimuli. Smac/DIABLO physically interacts with multiple IAPs and relieves their inhibitory effect on both initiator and effector caspases. Thus, Smac/DIABLO represents the mammalian functional homolog of the Drosophila Hid, Grim, and Reaper proteins. Recent structural studies reveal that the N-terminal tetrapeptide of Smac/DIABLO binds a surface groove on XIAP-BIR3, thus competitively removing the inhibition of caspase-9 by XIAP. Smac/DIABLO shares sequence homology with Hid, Grim, and Reaper only in the N-terminal 4 residues, prompting the hypothesis that Hid, Grim, and Reaper interact with DIAP1 using similar tetrapeptides and binding to a similar surface groove on DIAP1 (Wu, 2001 and references therein).
There is currently no structural information on DIAP1 or Hid, Grim, or Reaper. To investigate the structural mechanisms of DIAP1 recognition by the Drosophila Hid, Grim, and Reaper proteins, the DIAP1-BIR2 domain was crystalized by itself and in complex with the N-terminal peptides from both Hid and Grim (these structures were determined at 2.7, 2.7, and 1.9 Angstrom resolution, respectively). By analogy to the Smac-XIAP interactions, the first four amino acids of Hid and Grim bind an evolutionarily conserved surface groove on DIAP1-BIR2. The next 3 conserved residues of Hid and Grim also contribute to the interactions with DIAP1 through extensive van der Waals contacts. Interestingly, peptide binding to DIAP1-BIR2 appears to induce the formation of an additional alpha helix, which appears to stabilize peptide binding. In conjunction with biochemical analysis, this structural study reveals a molecular basis for the conservation and diversity necessary for the recognition of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins. These results have important ramifications for the design of IAP inhibitors toward therapeutic applications (Wu, 2001).
Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).
Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper that is detectably homologous to Grim and Hid is dispensable for Scythe binding (Thress, 1999).
Mammalian Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain. Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The Bruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. Drosophila Bruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1/Thread BIR2, be intact. Bruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that Bruce can regulate cell death at a novel point (Vernooy, 2002).
In Drosophila, the products of the reaper (rpr), head involution defective (hid), and grim genes are essential activators of caspase-dependent cell death. A genetic screen was carried out for suppressors of Rpr-, Hid-, and Grim-dependent cell death to identify regulators of their activity. Approximately 7000 new insertion lines of the GMREP P element transposon were generated. GMREP contains an engineered eye-specific enhancer sequence (GMR). This sequence is sufficient to drive the expression of linked genes in and posterior to the morphogenetic furrow during eye development. Thus, insertion of GMREP within a region can lead to the eye-specific expression of nearby genes. Each insertion line was crossed to flies that had small eyes due to the eye-specific expression of Rpr (GMR-Rpr flies), Hid (GMR-Hid flies), or Grim (GMR-Grim flies), and the progeny were scored for enhancement or suppression. A number of suppressors were identified. Five lines (GMREP-86A-1–5) mapped to the 86A region, and each strongly suppressed cell death induced by eye-specific expression of Rpr or Grim but not Hid. These lines mapped within a 6-kb interval. A number of other lines were obtained with P-element insertions located in the nearby region. Four of these, EP(3)0359, EP(3)0739, l(3)j8B6, and l(3)06142, mapped within six base pairs of the GMREP-86A-3–5 insertion sites. None of these, nor a fifth nearby line, l(3)06439, acted as a suppressor of GMR-Rpr-, GMR-Grim-, or GMR-Hid-dependent cell death. These results argue that the cell death suppression seen with the GMREP-86A lines was not due to a transposon-induced loss of function, but rather to the GMREP-dependent expression of a nearby gene. All of the GMREP-86A insertions were located 5' to a gene encoding the Drosophila homolog, Bruce, of murine Bruce (also known as Apollon in humans, suggesting this as an obvious candidate. The results of tissue in situ hybridizations with a Drosophila Bruce probe and immunocytochemistry with a Bruce-specific antibody support this possibility. Bruce transcript and protein are expressed at uniform low levels in wild-type eye discs. However, in the GMREP86A lines, they are expressed at high levels in and posterior to the morphogenetic furrow of the eye disc, which is where the GMR element drives expression (Vernooy, 2002).
To demonstrate that Bruce is responsible for the GMREP-86A-dependent suppression of Rpr- and Grim-dependent cell death, levels of the Bruce transcript were specifically downregulated in the eyes of flies carrying a GMR-Rpr transgene as well as a GMREP-86A element. Analysis focussed on one line, GMREP-86A-1, since all five lines behaved similarly with respect to cell death suppression and Bruce overexpression. Flies were generated that carried a Bruce RNA interference (RNAi) construct driven under GMR control (GMR-Bruce-RNAi flies). The eyes of GMR-Bruce-RNAi flies were normal. These animals were crossed to flies in which GMR-Rpr-dependent cell death was suppressed by the presence of the GMREP-86A-1 transposon and progeny from this cross were identified that carried all three transgenes, GMR-Bruce-RNAi, GMR-Rpr, and GMREP-86A-1. It was reasoned that if ectopic expression of Bruce in the eye, driven by the GMREP-86A-1 insertion, was responsible for the suppression of Rpr-dependent cell death, then expression of Bruce-RNAi should downregulate levels of the Bruce sense transcript. This should lead to an attenuation of the GMR-EP-86A-1-dependent suppression of Rpr-dependent cell death, causing a decrease in eye size. Such an attenuation was in fact observed. These observations, in conjunction with those obtained from studies with Bruce deletion mutants, argue that Bruce can suppress Rpr- and Grim-dependent cell death (Vernooy, 2002).
cDNAs encompasing the Bruce coding region were sequenced. This allowed an accurate map to be assembled of the Bruce exon-intron structure, which differs in some respects from that of the BDGP predicted gene. Overall, Bruce is 30% identical to murine Bruce. However, the Bruce N-terminal BIR domain and the C-terminal E2 domain show much higher degrees of homology, 83% and 86% identity, respectively. C. elegans homologs of Bruce were not apparent. Mutations in the Bruce gene were generated by carrying out imprecise excision of a P element, EP3731, located 3' to the Bruce transcript. Two deletions were generated that extended only in one direction, into the 3' end of the Bruce coding region. E12 deleted a relatively small region of the C terminus that includes the E2 domain, while E16 deleted approximately the C-terminal half of the Bruce coding region. Both lines were homozygous viable but male sterile. The possibility that E12 and E16 represent neomorphic mutations in Bruce cannot be excluded. However, the hypothesis that they represent hypomorphs or null mutations is favored, since they had the opposite phenotype of the GMREP-86A Bruce expression lines when in combination with GMR-Rpr, acting as enhancers rather than suppressors of Rpr-dependent cell death in the eye. E12 and E16 also enhanced GMR-Grim, but this effect is much more modest. E12 and E16 have no clear effect on cell death due to expression of Hid (Vernooy, 2002).
These results argue that endogenous Bruce levels, at least in the eye, are sufficient to act as a brake on Rpr-, and to some extent, Grim-dependent cell death. How does Bruce suppress apoptosis? A number of observations argue that Rpr- and Grim-dependent killing proceeds through distinct mechanisms and/or is regulated differently from those activities that is due to Hid. These differences are manifest at multiple points. At the level of DIAP1, point mutations of DIAP1 have effects on Rpr- and Grim-dependent cell death that are the opposite of those due to Hid. In addition, in a Drosophila extract, Hid, but not Rpr and Grim, promotes DIAP1 polyubiquitination. In contrast, in a different set of assays, Rpr and Grim, but not Hid, act as general inhibitors of protein translation. Finally, Rpr and Grim, but not Hid, show strong synergism with the effector caspase DCP-1 in terms of their ability to induce cell death in the eye. Each of these points defines a possible target for Bruce antiapoptotic action (Vernooy, 2002 and references therein).
Because Bruce strongly suppresses cell death induced by Rpr and Grim but not by Hid, one obvious possibility was that Bruce promotes Rpr and Grim ubiquitination and degradation. This hypothesis was tested by generating mutant versions of Grim and Rpr that lacked all lysines, the amino acid to which ubiquitin is added. These genes were introduced into flies under GMR control. GMR-Rpr-lys- and GMR-Grim-lys- flies have small eyes, indicating that these mutant proteins are effective cell death inducers. GMREP-86A-1-dependent Bruce expression suppresses this death very effectively, indicating that Bruce cannot be promoting ubiquitin-dependent degradation of Rpr or Grim. Interestingly, however, Bruce expression does not suppress cell death induced by expression of versions of Rpr (GMR-RprC) or Grim (GMR-GrimC) lacking their N termini, which are required for their IAP-caspase-disrupting interactions with the DIAP1 BIR2. This result is important because it argues that Bruce does not act to regulate this relatively uncharacterized death pathway (Vernooy, 2002).
The N-terminal Bruce BIR lacks a number of residues thought to be important for binding of Rpr, Hid, and Grim to DIAP1 BIR2. Thus, it seems unlikely that GMR-driven expression of Bruce inhibits cell death by simply titrating Rpr and Grim away from interactions with DIAP1 BIR2 as a result of similar interactions with the Bruce BIR. Nonetheless, the high degree of conservation between Bruce and mammalian Bruce in the BIR suggests that it is functionally important. To explore this role further, a fragment of Bruce that contained residues 1–531, including the BIR domain, was expressed under GMR control. Flies carrying this construct, GMR-Bruce-BIR flies, had normal appearing eyes, and in crosses to flies expressing GMR-Rpr, -Hid, or -Grim, GMR-Bruce-BIR did not enhance or suppress these eye phenotypes. These results do not rule out a role for the Bruce BIR in suppressing Rpr- and Grim-dependent cell death. However, they do suggest that the BIR alone is unlikely to mediate this inhibition (Vernooy, 2002).
Bruce overexpression in the eye also does not suppress cell death resulting from GMR-driven expression of the caspase Dronc, which is required for many apoptotic cell deaths in the fly, including those induced by expression of Rpr, Grim, and Hid. Dronc most resembles mammalian caspase-9, and its activation is likely to involve interactions with the Drosophila Apaf-1 homolog Ark. Thus, this result strongly suggests that Bruce does not block Ark-dependent Dronc activation or Dronc activity. This result is also suggested by the observation that decreasing Ark or Dronc in the eye strongly suppressed Hid-dependent cell death, which Bruce does not. A similar lack of cell death suppression is seen in the progeny of crosses between GMR-Bruce flies and flies expressing a second long prodomain caspase, Strica, whose mechanism of activation and normal functions are unknown. Finally, GMREP-86A-1 also fails to suppress the cell death due to GMR-dependent expression of the Drosophila proapoptotic Bcl-2 family member known variously as Debcl, Drob-1, dBorg-1, or Dbok (Vernooy, 2002).
Thus, the Bruce gene is found in mammals and flies, but not in the worm C. elegans. In humans, it is upregulated in some cell lines derived from gliomas and an ovarian carcinoma, and the results of antisense inhibition of Bruce suggest that it contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. The Drosophila homolog of Bruce, can potently inhibit cell death induced by Rpr and Grim but not Hid. In addition, flies with C-terminal deletions that removed the Bruce ubiquitin conjugation domain, or much larger regions of the coding region, acted as dominant enhancers of Rpr- and Grim-dependent, but not Hid-dependent, cell death. Together, these observations clearly demonstrate that Bruce can function as a cell death suppressor. The results with the deletion mutants suggest, but do not prove, that Bruce's death-inhibiting activity requires its function as a ubiquitin-conjugating enzyme. Based on the general conservation of cell death regulatory mechanisms, these results argue that mammalian Bruce is likely to facilitate oncogenesis by directly promoting cell survival in the face of specific death signals. One mechanism by which Rpr, Grim, and Hid promote apoptosis is by binding to DIAP1, thereby blocking its ability to inhibit caspase activity. It will be interesting to determine if mammalian Bruce also inhibits cell death induced by the expression of specific IAP binding proteins (Vernooy, 2002).
How does Bruce inhibit cell death? It does not promote the ubiquitination and degradation of Rpr and Grim directly. However, the possibility cannot be ruled out that Bruce somehow sequesters these proteins from their proapoptotic targets. The fact that it does not inhibit cell death due to Hid or Dronc expression argues that it is unlikely to be acting on core apoptotic regulators such as Ark, Dronc, or DIAP1, which are important for Hid-, Rpr-, and Grim-dependent cell death. An attractive hypothesis is that Bruce, perhaps in conjunction with apoptosis-inhibiting ubiquitin-protein ligases such as DIAP1 or DIAP2, promotes the ubiquitination and degradation of a component specific to Rpr- and Grim-dependent death signaling pathways. What might such a target be? Little is known about how Rpr- and Grim-dependent death signals differ from those due to Hid. However, one possibility is suggested by the recent observation that Rpr and Grim, but not Hid, can inhibit global protein translation. This creates an imbalance between levels of short-lived IAPs and the caspases they inhibit, thereby sensitizing cells to other death signals. Perhaps Bruce targets a protein(s) required for this activity (Vernooy, 2002).
Finally, Bruce is a very large protein, and thus its coding region might be expected to be subject to a relatively high frequency of mutation. Truncation of Bruce through the introduction of a stop codon or a frame shift is thus likely to be a relatively common form of Bruce mutation. The results of the deletion analysis show that C-terminal Bruce truncations act to enhance cell death in response to several different signals. Given this, it will be interesting to determine if human Bruce mutations are associated with a predisposition to pathologies that involve an inappropriate increase in cell death (Vernooy, 2002).
Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. Voltage-dependent Shaker (Sh) K+ channels open in response to depolarization and subsequently undergo N-type inactivation by a "ball and chain" mechanism. The 20 N-terminal residues of the ShB channel form the inactivation "ball," which is tethered to membrane-spanning channel domains by the following ~200-residue "chain." Inactivation occurs when the N-terminal inactivation ball physically occludes the inner pore of the channel from the cytoplasmic side. Stability of the inactivated state is enhanced by the hydrophobicity of approximately the first 10 residues of the inactivation ball, whereas positively charged amino acids within the following 10 residues promote entry into the inactivated state via electrostatic interactions. Deletions in the distal N terminus of the channel disrupt inactivation, which can be reversed by application of a 20-residue synthetic peptide corresponding to the initial N-terminal sequence of the channel. Ancillary beta subunits in some K+ channel complexes serve to produce N-type inactivation by a similar mechanism. The conserved N-terminal sequences of Reaper, Grim, and Hid resemble those N-terminal Sh K+ channel domains that are involved in inactivation. This sequence similarity led to the hypothesis that Reaper, Grim, and Hid facilitate initiation of apoptosis by inducing inactivation of K+ channels. Sustained inactivation of K+ channels will result in chronic membrane depolarization that may lead to the initiation of the caspase-dependent apoptotic program, perhaps by increasing the level of cytosolic free Ca2+. Synthetic Reaper and Grim N terminus peptides are shown to induce fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that is qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlates with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein causes near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels (Avdonin, 1998).
Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It has not been clear how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. This study found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in a reconstitution assay. However Grim does not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2 (Yoo, 2005).
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