Relish : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Relish

Synonyms -

Cytological map position - 85C4--6

Function - transcription factor

Keywords - immune response

Symbol - Rel

FlyBase ID: FBgn0014018

Genetic map position - 3-

Classification - Ankyrin-repeat and NFkappaB domain protein

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Sudmeier, L.J., Samudrala, S.S., Howard, S.P. and Ganetzky, B. (2015). Persistent activation of the innate immune response in adult Drosophila following radiation exposure during larval development. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26333838
Summary:
This study investigates the role of the innate immune system in response to radiation exposure. It was shown that the innate immune response and NF-ĸB target gene expression is activated in the adult Drosophila brain following radiation exposure during larval development and that this response is sustained in adult flies weeks after radiation exposure. Preliminary data suggest that innate immunity is radioprotective during Drosophila development. Together these data suggest that activation of the innate immune response may be beneficial initially for survival following radiation exposure but result in long-term deleterious consequences, with chronic inflammation leading to impaired neuronal function and viability at later stages. This work lays the foundation for future studies of how the innate immune response is triggered by radiation exposure and its role in mediating the biological responses to radiation.
Ji, Y., Thomas, C., Tulin, N., Lodhi, N., Boamah, E., Kolenko, V. and Tulin, A. V. (2016). Charon mediates immune deficiency-driven PARP-1-dependent immune responses in Drosophila. J Immunol [Epub ahead of print]. PubMed ID: 27527593
Summary:
Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. This article describes a novel molecular mechanism controlling NF-κB-dependent innate immune response. A previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila. Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. The results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, these results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.
Morris, O., Liu, X., Domingues, C., Runchel, C., Chai, A., Basith, S., Tenev, T., Chen, H., Choi, S., Pennetta, G., Buchon, N. and Meier, P. (2016). Signal integration by the IkappaB protein Pickle shapes Drosophila innate host defense. Cell Host Microbe 20: 283-295. PubMed ID: 27631699
Summary:
Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. This study describes Pickle (CG5118), a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-kappaB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.
Tavignot, R., Chaduli, D., Djitte, F., Charroux, B. and Royet, J. (2017). Inhibition of a NF-κB/Diap1 pathway by PGRP-LF is required for proper apoptosis during Drosophila development. PLoS Genet [Epub ahead of print]. PubMed ID: 28085885
Summary:
NF-κB pathways are key signaling cascades of the Drosophila innate immune response. One of them, the Immune Deficiency (IMD) pathway, is under a very tight negative control. Although molecular brakes exist at each step of this signaling module from ligand availability to transcriptional regulation, it remains unknown whether repressors act in the same cells or tissues and if not, what is rationale behind this spatial specificity. This study shows that the negative regulator of IMD pathway PGRP-LF is epressed in ectodermal derivatives. In the absence of any immune elicitor, PGRP-LF loss-of-function mutants display a constitutive NF-κB/IMD activation specifically in ectodermal tissues leading to genitalia and tergite malformations. In agreement with previous data showing that proper development of these structures requires induction of apoptosis, it was found that ectopic activation of NF-κB/IMD signaling leads to apoptosis inhibition in both genitalia and tergite primordia. NF-κB/IMD signaling antagonizes apoptosis by up-regulating expression of the anti-apoptotic protein Diap1. Altogether these results show that, in the complete absence of infection, the negative regulation of NF-κB/IMD pathway by PGRP-LF is crucial to ensure proper induction of apoptosis and consequently normal fly development. These results highlight that IMD pathway regulation is controlled independently in different tissues, probably reflecting the different roles of this signaling cascade in both developmental and immune processes.

He, X., Yu, J., Wang, M., Cheng, Y., Han, Y., Yang, S., Shi, G., Sun, L., Fang, Y., Gong, S. T., Wang, Z., Fu, Y. X., Pan, L. and Tang, H. (2017). Bap180/Baf180 is required to maintain homeostasis of intestinal innate immune response in Drosophila and mice. Nat Microbiol 2: 17056. PubMed ID: 28418397
Summary:
Immune homeostasis is a prerequisite to protective immunity against gastrointestinal infections. In Drosophila, immune deficiency (IMD) signalling (tumour necrosis factor receptor/interleukin-1 receptor, TNFR/IL-1R in mammals) is indispensable for intestinal immunity against invading bacteria. However, how this local antimicrobial immune response contributes to inflammatory regulation remains poorly defined. This study shows that flies lacking intestinal Bap180 (a subunit of the ]SWI/SNF complex) are susceptible to infection as a result of hyper-inflammation rather than bacterial overload. Detailed analysis shows that Bap180 is induced by the IMD-Relish response to both enteropathogenic and commensal bacteria. Upregulated Bap180 can feed back to restrain overreactive IMD signalling, as well as to repress the expression of the pro-inflammatory gene eiger (TNF), a critical step to prevent excessive tissue damage and elongate the lifespan of flies, under pathological and physiological conditions, respectively. Furthermore, intestinal targeting of Baf180 renders mice susceptible to a more aggressive infectious colitis caused by Citrobacter rodentium. Together, Bap180 and Baf180 serve as a conserved transcriptional repressor that is critical for the maintenance of innate immune homeostasis in the intestines.
Maki, K., Shibata, T. and Kawabata, S. I. (2017). Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish. J Biol Chem [Epub ahead of print]. PubMed ID: 28258224
Summary:
In Drosophila, the immune deficiency (IMD) pathway-dependent signal is finally transmitted through proteolytic conversion of the nuclear factor-κB-like transcription factor Relish to the active N-terminal fragment Relish-N to induce its translocation from the cytosol into the nucleus for the expression of IMD-controlled genes. Previous studies have demonstrated that transglutaminase (TG) suppresses the IMD pathway by polymerizing Relish-N to inhibit its nuclear translocation. A synthetic amine, such as monodansylcadaverine (DCA) or biotin-labeled pentylamine, ingested by flies is TG-dependently incorporated into Relish-N, causing the nuclear translocation of modified Relish-N in gut epithelial cells. DCA-incorporated Gln residues were located in the Rel homology domain, the DNA-binding region of Relish-N. TG-catalyzed DCA incorporation inhibits the binding of Relish-N to the Rel-responsive element of the κB sequence. TG localizes not only in the cytosol but also in the nucleus. Natural polyamines, including spermidine and spermine, competitively inhibited TG-dependent DCA incorporation into Relish-N. Relish-N is also modified by spermine, and the transcription of cecropin A1 and diptericin genes controlled by the IMD pathway is reduced. These findings suggest that intracellular TG regulates the transcriptional activity of Relish-N through the incorporation of polyamines into Relish-N as well as through protein-protein crosslinking of Relish-N.
BIOLOGICAL OVERVIEW

Relish was identified in a screen for genes involved in the Drosophila immune system by using PCR differential display to identify genes induced in infected flies. Unlike the single domain proteins Dorsal and Cactus (respectively the classic NF-kappaB- and IkappaB-related proteins of Drosophila), Relish contains both a Rel homology domain (hence the name Relish) and an IkappaB-like domain with six ankyrin repeats. Thus Relish is a dual domain protein. In this respect Relish is similar to the compound mammalian NF-kappaB precursors p100 and p105, although no obvious similarity is seen outside the two conserved domains (Dushay, 1996).

Interesting features have been noted in the regions outside the conserved domains. Like RelB, Relish has an unusually long region N-terminal of the Rel homology domain. Furthermore, just downstream of the Rel homology domain there is a serine-rich stretch, corresponding to the position where p100 and p105 have a glycine-rich region that serves as a processing signal for the generation Rel domain containing p50. The serine-rich sequence in Relish may serve a similar function. Another serine-rich region is found in the N-terminal region of Relish. Finally, there are several potential target sites for phosphorylation by casein kinase II, including four in the spacer between the Rel and ankyrin domains, and five either in or near the PEST region. Casein kinase II has been implicated in the constitutive phosphorylation of the PEST region in IkappaB, and the signal-induced phosphorylation of the same protein is mediated by a kinase with similar target sites (Dushay, 1996).

There is experimental evidence for at least three independent signal transduction pathways in the activation of the immune response in Drosophila. One pathway, defined by the Toll gene, is involved in the defense against fungi. A constitutively active form of Toll can mediate superinduction of the Cecropin gene promoter in the transfected hemocyte line mbn-2. Toll is preferentially involved in the induction of antifungal components such as Drosomycin and metchnikowin. The Toll pathway also requires other dorsal group genes such as tube, pelle, cactus, and spatzle (spz), but not dorsal itself. A second pathway directs an antibacterial response. It is defined by the immune deficiency (imd) mutation, which affects the expression of diptericin and other bactericidal factors, but not of drosomycin. In agreement with this model, factors that have both antibacterial and antifungal activity, such as the cecropins and metchnikowin, appear to respond to both pathways. In addition to these two pathways, recent experiments with the Toll-like gene, 18-wheeler, indicate that a third pathway may exist. Mutations in 18-wheeler affect the induction of Attacin, but not Diptericin or Drosomycin (Hedengren, 1999 and references therein).

Although one or more Rel factors are likely to mediate signals from these pathways in the immune response, the specific role of the three known Rel factors in Drosophila is unclear. Dorsal is not required, because dorsal mutants have no phenotypic effect on the induction of the antimicrobial genes. However, recent data show that Dif is specifically involved in the induction of drosomycin and that Dorsal may play a redundant role in the same pathway. Using P element-mediated mutagenesis, a series of deletions in the Relish gene have been created. These mutations have a profound effect on the induction of the entire set of antimicrobial genes, and it is concluded that Relish is a key factor that controls the antibacterial as well as the antifungal defense in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three immunity pathways of Drosophila (Hedengren, 1999)

Relish is a key factor in the induction of the humoral immune response in Drosophila, including antibacterial as well as antifungal factors. In striking contrast, there is a complete lack of effect of Relish on cellular immune reactions and hematopoiesis. These observations indicate that cellular and humoral immune responses are controlled by distinct and independent systems in Drosophila. Hematopoiesis, and perhaps the activation of cellular reactions, may instead be controlled by another Rel factor, Dorsal. Overexpression of this factor has been shown to induce lamellocyte differentiation and cause the formation of melanotic capsules, and similar effects have been observed with mutations that lead to the activation of the Toll pathway (Hedengren, 1999 and references therein).

It is interesting to compare the roles of different Rel factors in the cellular and humoral immune responses of Drosophila with the situation in mammals. A parallel can be drawn between the phenotype of the Relish mutants and the effects seen in knockout mice that carry a disruption of the p105 gene. Such mice have a normal complement of lymphocytes, but they are deficient in both specific and nonspecific responses to infection (Sha, 1995). In contrast, disruption of the RelA and RelB genes in mice gives more complex developmental phenotypes, including effects on hematopoiesis. Thus, although the picture is less clear in mice than in Drosophila, it appears to be a conserved feature that different Rel factors regulate immune responses and hematopoiesis (Hedengren, 1999 and references therein).

The relative importance of the humoral factors in Drosophila immunity is dramatically illustrated by the phenotype of the Relish mutants. A single bacterial cell is sufficient to kill these mutants and the resistance to fungal infections is also impaired, even though the cellular immune defense appears to be intact and parasites are efficiently eliminated. This extreme sensitivity to a bacterial infection is surprising, since phagocytosis of bacteria appears to be normal. One possible explanation is that the antibacterial effector functions of the phagocytic cells are impaired in the Relish mutants. Alternatively, and perhaps more likely, enterobacteria such as Enterobacter cloacae may be relatively resistant to the killing mechanisms of the hemocytes. Indeed, injected cells of Enterobacter cloacae or Escherichia coli can survive for a long time in wild-type Drosophila, even though bacteria injected later are eliminated efficiently by the induced immune response. The surviving bacteria probably reside within phagocytic cells, where they are protected from the humoral factors. Thus, the humoral immune response may be the only efficient defense against bacteria such as Enterobacter cloacae or Escherichia coli (Hedengren, 1999 and references therein).

In spite of the dramatic effects on the immune response, homozygous Relish mutants are fertile and give rise to normal offspring. This lack of Relish developmental effects is surprising, considering the fact that early embryos express a specific, maternally contributed form of Relish that lacks the N-terminal domain (Dushay, 1996). The existence of a specific embryonic form suggests that Relish, like dorsal, could play a role in embryogenesis. However, the function of Relish in the early embryo, if any, must be redundant or else it is involved in a more subtle process that is not required for survival (Hedengren, 1999).

Two alternative models are suggested for the role of Relish in the three genetically defined induction pathways of immune responses in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three pathways. The case is most clear for the imd pathway, which is of particular importance for the induction of Diptericin and other genes of the antibacterial defense. Since Diptericin induction has an absolute requirement for a functional Relish gene, it has been concluded that Relish is likely to be part of this pathway. Furthermore, Relish could also be involved in the Toll and 18-wheeler pathways, since the inducibility of both Drosomycin and Attacin is significantly reduced in Relish mutants. However, additional factors have to be invoked in order to explain how different pathways can specifically induce different genes. For the Toll pathway, this specificity could be provided by Dif and/or dorsal since these factors have been shown to participate in the Toll-dependent induction of Drosomycin, perhaps in the form of a Relish-Dif (or Relish-Dorsal) heterodimer. In this case, a homo- or a hetero-dimer of Dif and/or Dorsal could partially substitute for loss or Relish, explaining the residual expression of Drosomycin in the Relish mutants. Data supporting this model were very recently obtained by Han (1999). A synergistic effect on Drosomycin expression was found when Relish was overexpressed together with Dif. This indicates that the two factors may indeed cooperate, perhaps as a heterodimer, in the Toll pathway. For the other pathways, no candidates for such specificity factors have been found. The induction of Drosomycin and Attacin is not completely abolished in the Relish mutants, and for this reason at least one Relish-independent pathway must be included in the model (Hedengren, 1999 and references therein).

A second model, which is also consistent with these findings, is suggested by the fact that there is a residual inducibility of Drosomycin in Toll mutants and of Attacin in 18-wheeler mutants. According to this model, Relish and Toll (the latter probably via Dif) act independently to induce Drosomycin, and similarly Relish and 18-wheeler form independent pathways for the induction of Attacin (Hedengren, 1999 and references therein).

It is interesting to observe the complexity of the different signal transduction pathways that converge to activate the immune responses in Drosophila. At least two of these pathways depend on receptors of the Toll family. Several other members of this family can be identified in the database of the Drosophila genome project, and it is possible that yet other signal pathways will be identified as the role of these receptors becomes clear. The availability of Relish and other mutants will help in understanding these different pathways and their role in immunity, not only in Drosophila but probably also in humans (Hedengren, 1999).

Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes

The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase [consisting of 2 subunits: a catalytic kinase subunit encoded by ird5 (IKKβ) and a regulatory subunit encoded by kenny (IKKγ)] and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. This report shows that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish (Ertürk-Hasdemir, 2009).

These data suggest a new model for the regulation of Relish activity. In this model, Relish is controlled by 2 distinct mechanisms, both of which signal downstream of the receptor Peptidoglycan recognition protein LC (PGRP-LC). One arm controls the cleavage of Relish and requires IMD, FADD, DREDD, and the IKK complex. The other arm controls Relish phosphorylation through TAK1 and the IKK complex. Robust induction of antimicrobial peptide expression requires that both mechanisms of control are fully active; Relish must be cleaved and phosphorylated (Ertürk-Hasdemir, 2009).

Phosphorylation of Relish is critical for signal-dependent transcriptional activation of target genes. By using mass spectrometry and in vitro kinase assays, serines 528 and 529 were identified as targets of IKKβ phosphorylation. These serines are phosphorylated rapidly, in cell lines and flies, after immune challenge. Mutation of these residues to alanine resulted in a protein that acted as dominant negative in cell culture, inhibiting the PGN-induced expression of antimicrobial peptide genes Diptericin and Attacin. A recent study reported that ectopic expression of REL-68 (the N-terminal portion of Relish) was not sufficient to drive the expression of the antimicrobial peptide genes Attacin and Cecropin (Wiklund, 2009). Because REL-68 is not expected to be phosphorylated, these results support the conclusion that phosphorylation is critical for Relish-mediated transcriptional activation of AMP genes. However, this article also reported that REL-68 was able to induce Diptericin, which appears to contradict the current findings. In these experiments, transgenic REL-68 is overexpressed, which may contribute to these confusing results (Ertürk-Hasdemir, 2009).

Further supporting the conclusion that Relish phosphorylation is critical, kinase-dead IKKβ transgenic rescue supported only very weak induction of AMP genes in flies. In these experiments, Relish is expressed at normal levels. Surprisingly, serines 528 and 529, and IKKβ catalytic activity itself, were not required for signal-dependent Relish cleavage. Serines 528 and 529 were also not essential for nuclear translocation or DNA binding. Instead, ChIP experiments show that these serines are required for the efficient recruitment of RNA Pol II to the Diptericin locus (Ertürk-Hasdemir, 2009).

These ChIP assays used the 8WG16 mAb, which preferentially recognizes unphosphorylated CTD repeats of the largest subunit of RNA Pol II. The unphosphorylated CTD is associated with the preinitiating RNA Pol II complex recruited to promoters. Thus, these results argue that phosphorylation of Relish on serines 528 and 529 is required for efficient recruitment of RNA Pol II to the Diptericin and Diptericin-B promoters. An alternate possibility, suggested by recent findings on gene regulation in Drosophila, is that phosphorylated Relish could stimulate elongation from paused RNA Pol II. However, a genome-wide analysis of promoters containing stalled RNA Pol II has found that many Drosophila antimicrobial peptide genes are not sites of paused RNA Pol II. The use of 8WG16 in the experiments presented in this study, further argues that phosphorylation of serines 528 and 529 does not modulate RNA Pol II pausing but instead regulates polymerase recruitment to the preinitiation complex at the Diptericin locus. The exact mechanism by which phosphorylation of serines 528 and 529 affect RNA Pol II recruitment remains to be elucidated. It may involve interaction with coactivators, such as components of the mediator complex, or it may involve the recently discovered IMD component Akirin (Goto, 2008), which is argued to function in the nucleus, downstream of Relish (Ertürk-Hasdemir, 2009).

This report also provides further supporting evidence that DREDD may be the caspase that directly cleaves Relish. This study shows that overexpression of DREDD is sufficient to cause Relish cleavage. Relish cleavage required catalytically active DREDD and expression of another apical caspase, the caspase-9 like DRONC, did not generate cleaved Relish. Interestingly, DREDD-mediated Relish cleavage did not lead to Relish phosphorylation and was not sufficient to drive Diptericin expression. Furthermore, immunopurified DREDD, but not drICE, cleaved Relish in vitro, albeit not very efficiently. The poor efficiency of Relish cleavage, in vitro, may be due to the highly oligomeric state of purified Relish and/or the low activity of DREDD, which has proven to be very difficult to produce in an active form. It was also found that a biotinylated peptide with the Relish cleavage site bound active DREDD; although strong evidence for a direct interaction, this assay is not particularly specific. Together, these data strongly suggest that DREDD directly cleaves Relish, but it cannot yet be concluded with certainty that other proteases, such as an effector caspase, are not involved (Ertürk-Hasdemir, 2009).

In addition to DREDD, Relish cleavage also requires both IKK subunits. However, Relish cleavage does not require catalytically active IKKβ. Delaney (2006) showed that TAK1 is not required for Relish cleavage. Because TAK1 is required for the immune-induced activation of the IKK kinase, this result is consistent with the data indicating that IKK catalytic activity is not involved in Relish cleavage. Instead, IKK complex may function as a scaffold or adaptor, but not as a kinase, in controlling the cleavage of Relish (Ertürk-Hasdemir, 2009).

Taken together, these data demonstrate that Relish is regulated by 2 distinct mechanisms. Relish is probably cleaved by DREDD and phosphorylated by the IKK complex. These 2 regulatory mechanisms appear to be independent, because phosphorylation can occur without cleavage, and vice versa, although they are both triggered by PGN stimulation of the receptor PGRP-LC. Surprisingly, the IKK complex also plays a role in the cleavage of Relish, but not through its kinase activity. Instead, IKK-mediated phosphorylation of Relish on serines 528 and 529, within its N-terminal transcription factor module, is necessary for transcriptional activation of target genes (Ertürk-Hasdemir, 2009).


GENE STRUCTURE

All three Relish transcripts contain both Rel and ankyrin domains, since hybridization of Northern blots with probes from both domains show identical results. To further characterize the maternal transcript, five cDNA clones were isolated from an ovarian library. These clones are identical to those from the adult and mbn-2 cell libraries except for various degrees of truncation at the 5' end. The longest clone has an insert of 2.7 kb and thus must be near full-length. 5'-rapid amplification of cDNA end products from 0- to 2-h-old embryos show the same sequence and terminate near the 5' end of this clone. It is concluded that the maternal transcript differs from the other two Relish transcripts at the 5' end only, and that if there is a unique 5' exon it must be very short. The maternal transcript is too short to encode the N-terminal part of the open reading frame, and has an alternative translation start site (Dushay, 1996).

Relish maps near the end of the 85C region on the right arm of the third chromosome. This is a well-characterized region of the genome, covered by contiguous genomic walks around the oskar and pumilio genes, as well as by genomic clones around neuralized. Cosmid clones were obtained from the relevant interval and they were probed with the Relish cDNA clone 5.3 (Dushay, 1996). In this way, Relish could be mapped to a cosmid, cos 1698, between pumilio and neuralized. 10.8 kb around the Relish gene were sequenced and several cDNA clones corresponding to the transcripts of this region were isolated. The genomic organization in this region turns out to be complex, with overlapping transcripts from at least four different genes. Twenty cDNA clones from the Relish gene have been described (Dushay, 1996). They are all collinear, and there is no evidence for alternative splicing of the Relish gene or for the use of alternative polyadenylation sites. Nevertheless, at least three different Relish transcripts of 2.7, 3.1, and 3.4 kb can be detected on Northern blots (Dushay, 1996). The 2.7 kb transcript is expressed in females and in early embryos, presumably as a maternally contributed transcript. This conclusion is supported by the observation that Relish is expressed in nurse cells in the ovary, as detected by in situ hybridization (Hedengren, 1999).

The 3.1 kb transcript is very strongly induced in infected flies, whereas the 3.4 kb transcript is constitutively present. 5' RACE has been used to sequence and accurately map the 5' ends of these transcripts, as well as of a fourth, 3.5 kb transcript of low abundance. The latter transcript can also occasionally be detected as a faint band on Northern blots. These data show that the different Relish transcripts originate from one minor and three major transcription start sites that are arranged in tandem. The entire Relish transcription unit is situated inside an intron of the previously described Nmdmc gene (Price, 1993), which encodes a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (Hedengren, 1999).

This is a bifunctional enzyme involved in tetrahydrofolate metabolism, with no obvious relationship to immune responses. From the Nmdmc gene, four different cDNA clones have been isolated, all of them representing the same splice form (transcript A) with a first exon 2.4 kb upstream of Relish, spliced to two exons downstream of the Relish gene. The first exon of this transcript differs from that of the previously published Nmdmc transcript (transcript B; Price, 1993) for which the first exon could be mapped to a position 1.2 kb upstream of Relish. Thus, the Nmdmc gene has two alternative splice forms, both of them spanning the region that contains the four Relish transcripts. The protein products encoded by the two transcripts are essentially identical. In transcript B, the first exon is noncoding, whereas transcript A encodes an additional six N-terminal amino acid residues (Hedengren, 1999).


PROTEIN STRUCTURE

Amino Acids - 817

Structural Domains

Relish contains both a Rel homology domain and an IkappaB-like domain with six ankyrin repeats. A phylogenetic reconstruction of the possible relationship between the different Rel homology domains shows that Relish may have branched off from other Rel proteins at a very early stage. The sequences of the ankyrin repeats are also quite different from those of other IkappaB-like proteins, and they are about equally close to the ankyrin repeats in the Notch, ankyrin and IkappaB families. As in other Rel proteins, a putative nuclear localization sequence is found at the C-terminal end of the Rel homology domain, and the ankyrin repeats are followed by an acidic, PEST-like sequence. PEST sequences are rich in proline, glutamic acid, serine and threonine residues, and they have been implicated in protein turnover (Dushay, 1996).


Relish : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 November 2000

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