hephaestus: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - hephaestus

Synonyms -

Cytological map position - 100C2--4

Function - RNA-binding protein

Keywords - Notch pathway, wing, mRNA splicing, spermatogenesis

Symbol - heph

FlyBase ID: FBgn0011224

Genetic map position - 3R

Classification - RNA recognition motif, polypyrimidine tract binding protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Recent literature
Sridharan, V., Heimiller, J., Robida, M. D. and Singh, R. (2016). High throughput sequencing identifies misregulated genes in the Drosophila polypyrimidine tract-binding protein (hephaestus) mutant defective in spermatogenesis. PLoS One 11: e0150768. PubMed ID: 26942929
Summary:
The Drosophila polypyrimidine tract-binding protein (dmPTB or Hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. This study has identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. This analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. It was shown, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB/Hephaestus and that the site is conserved in many Drosophila species. Thus Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

Drosophila hephaestus (heph) is required to attenuate Notch activity after ligand-dependent activation during wing development. The original male sterile heph allele was identified in a genetic screen for loci required for spermatogenesis (Castrillon, 1993). New lethal alleles of heph have been isolated that affect wing margin and wing vein pattern formation in genetic mosaics. The Drosophila heph gene encodes the apparent homolog of mammalian polypyrimidine tract binding protein (PTB). PTB was first identified in vertebrates as a protein that binds to intronic polypyrimidine tracts preceding many 3' pre-mRNA splice sites (Garcia-Blanco, 1989). Many different functions have been identified for vertebrate PTB, including the control of alternative exon selection (Carstens, 2000; Chan, 1997; Chou, 2000; Cote, 2001; Lou, 1999; Mulligan, 1992; Perez, 1997; Southby, 1999; Zhang, 1999), translational control or internal ribosome entry site (IRES) use (Hunt, 1999; Ito, 1999; Kim, 2000; Pilipenko, 2000), mRNA stability (Tillmar, 2002) and mRNA localization (Cote, 1999). PTB may also act as a transcriptional activator (Rustighi, 2002). The study of heph is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in Notch signaling (Dansereau, 2002 and references therein).

Somatic clones lacking heph express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of Delta. These results led to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta (Dansereau. 2002).

Ectopic Notch activation is sufficient to induce margin formation in adjacent tissue in any wing compartment without affecting the compartment identity of those cells. This similarity in ectopic margin phenotypes and the expression of the Notch target genes wg and cut in heph clones suggests that heph acts as a repressor of Notch activation. Inhibition of vein differentiation is also a characteristic phenotype of ectopic activation of the Notch signaling pathway. Autonomous loss of vein differentiation in heph clones is the same phenotype observed in clones of Hairless, which encodes a transcriptional co-repressor of Notch target gene expression. Vein loss is also characteristic of gain-of-function Abruptex alleles of Notch, ectopic expression of the genes of the E(Spl)-C, or expression of low levels of the activated form of Notch (Dansereau. 2002).

Direct evidence of Notch activation was also observed in heph mutant clones situated throughout the wing imaginal disc. Increased levels of the Notch intracellular domain (NICD) were found in the cell body of normal boundary cells and in heph mutant cells. Although it is not known that these changes represent genuine nuclear accumulation, the altered distribution of NICD, which is absent in Su(H) mutant tissue, is consistent with models of Notch activation involving Su(H)-mediated translocation of NICD to the nucleus. The position independence of this effect indicates that heph acts in all wing disc cells to modulate Notch pathway function and is consistent with reports that low levels of Notch pathway activation are required throughout the developing disc for cell proliferation and for the weak general expression of Notch target genes such as E(spl)mß (Dansereau. 2002).

Although the effects of heph on Dl, Ser and NICD levels are position independent, the margin-inducing effects of heph are restricted to a competent region within a few cell diameters of the endogenous wing margin. By contrast, clones of cells expressing NICD under the control of a strong promoter are able to induce wg and cut expression, as well as ectopic margin and outgrowths throughout the wing blade, independent of the position of the clone. The effects of heph clones, including vein loss and the restricted location of ectopic margin, more closely resemble the effects reported for clones expressing NICD under a weak promoter. The competent region corresponds roughly to the domain of Dl and Ser expression in the regions flanking the boundary. As defined by the refinement of wg expression, cells in this region are initially Notch activated and express wg during late second and early third instar. The flanking cells subsequently repress Notch activation and lose wg expression in response to increased levels of Dl and Ser. It is likely that the band of cells that maintains wg expression begin with higher levels of Notch activation and that this bias is what allows them to maintain Notch activation and wg expression. Even small increases in Notch activity caused by the loss of heph activity in the cells of the flanking domains may bias the signaling required to refine the wg and Dl/Ser domains in favor of maintaining Notch activity in heph clones. Thus, the competent region for heph-induced margin formation may reflect the high levels of Notch activation required for margin induction (Dansereau. 2002).

The maintenance of Notch activation in heph cells in the flanking regions may cause them to become more like boundary cells. Thus, they maintain wg expression and decrease Dl and Ser expression. The reduction in Dl/Ser in heph clones would reduce Notch inhibition to allow further increases in Notch activation by adjacent Dl/Ser-expressing cells. This is consistent with the induction of ectopic cut expression, which only in heph mutant cells abutting the Dl/Ser domain may require the highest levels of Notch activation. A bias toward Notch activation in heph mutant cells may also explain the unusual shape of the boundary cell domain that is often observed in heph mosaic discs. Boundary Notch activation is required to maintain the lineage restriction property of the DV boundary and perturbing the spatial pattern of Notch activation can alter the shape of the boundary (Dansereau. 2002).

It has been reported that Notch activation increases Dl and Ser expression in the wing pouch. However, these effects are due to a positive feedback loop mediated indirectly through wg. Cells mutant for heph located away from the wing margin do not activate wg expression and do not activate Dl and Ser expression, perhaps because of insufficient levels of Notch activation. Instead, an autonomous decrease is observed in Dl and Ser expression throughout heph clones. The decrease in Dl expression depends on Su(H), and by implication is downstream of Notch activity in heph mutant cells. These observations are consistent with genetic evidence that Notch activity represses Delta activity, and with instances of Notch signaling that require the progressive restriction of ligand and receptor to reciprocal cells (e.g. during wing margin and wing vein patterning (Dansereau. 2002).

Is heph specific to the Notch signaling pathway? It is unlikely that the Drosophila PTB homolog (heph) is a dedicated Notch pathway component considering that several target RNAs not involved in Notch signaling have been identified for vertebrate PTBs (Valcarcel, 1997; Wagner, 2001). This is especially clear when considering heph-induced wing margin nicks and the under-proliferation or lack of survival of heph mutant cells, neither of which are Notch gain-of-function phenotypes. Both of these defects may result from the disruption of heph targets required for cell survival. Strong decreases in cell survival in clones would mask the enhancement of proliferation provided by Notch activation. Cell death in clones in the wing margin could result in wing margin nicks through nonspecific disruption of wg expression at the DV boundary. This may explain the greater recovery of nicked wings resulting from clones induced prior to the formation of the DV boundary, which are able to occupy both the dorsal and ventral surfaces of the wing blade (Dansereau. 2002).

In addition to the heph wing phenotypes discussed above, the effects of heph mutations have been observed on other imaginal tissues, and clearly heph affects some but not all Notch dependent development. For example, heph clones cause ommatidial pattern defects, whereas little or no effect is observed in the formation of leg joints or in the development of thoracic microcheatae. This suggests that the Notch pathway requires modification to accommodate the diversity of processes it regulates and that, as in the case of modifiers such as numb and Suppressor of deltex, heph may be essential for only a limited subset of Notch signaling events. The specificity of heph phenotypes also argues against Notch activation in heph clones being due to the amplification of generic defects in signal transduction or transcription as a result of the exquisite dosage sensitivity of Notch signaling. Indeed, the lateral inhibition signaling that regulates the spacing of adult thoracic microcheatae, perhaps the most dosage-sensitive Notch process, is relatively unaffected by loss of heph activity. Thus, although heph is unlikely to be dedicated to the Notch signaling pathway, it is most likely to play a specific role in the regulation of some Notch signaling events (Dansereau. 2002).

How does heph regulate Notch activity? This study has linked together for the first time the PTB/hnRNPI RNA-binding proteins and the Notch signaling pathway. Given the strong sequence similarity shared between heph and vertebrate PTBs, it is probable that heph regulates the processing, stability or translation of a Notch pathway mRNA. However, the heph mosaic wing phenotypes most closely resemble the effects of low level ectopic Notch activation and cannot be easily correlated with an effect on any particular known element in the Notch pathway. The phenotypes of clones mutant for Delta and heph are most informative in explaining where heph acts in the Notch pathway. The epistasis of Dl over heph in double mutant clones indicates that the Notch activation in heph clones depends on Dl. This ligand dependency excludes the possibilities that Notch target genes are generally de-repressed, or that the Notch receptor is constitutively activated, in heph mutant cells. Rather, it suggests that in the absence of heph, existing Notch activity is amplified and/or maintained. Therefore, the favoured explanation is that heph is required to attenuate Notch activity after ligand-dependent activation (Dansereau. 2002).

The phenotypic consequences of heph are most prominent in the wing margin cells and wing vein cells. Both of these cell types require decreases in the levels of Notch activity during development and the heph phenotype results from persistent Notch activity in these cells. The wing margin cells lose Notch activation and wg expression during the refinement of wg and Dl/Ser expression during the late second and early third instar. During larval development, the cells that will ultimately give rise to the vein express low levels of Notch and Notch target genes such as E(spl)mß, indicating that these cells have low levels of Notch activation prior to the repression of Notch transcription in pupal vein cells. Although it is not certain how these cells normally lose Notch activation, one possibility is that NICD stability is tightly regulated in order for cells to change Notch activation states and that heph+ may be required for cells to degrade NICD following ligand activation of the Notch receptor (Dansereau. 2002).

Several lines of evidence suggest that regulated degradation of NICD may be crucial. NICD includes a PEST domain, a characteristic of proteins with very short half-lives, and mutations of Notch that delete the PEST domain are associated with N(gf) phenotypes, albeit in different Notch signaling events than those affected by heph. The ubiquitin-proteasome pathway has been implicated in regulating the degradation of a NICD related protein in C. elegans, where the Notch family receptor LIN-12 is negatively regulated by sel-10. The C. elegans and mammalian SEL-10 proteins both contain F-box motifs, and are thought to form part of a ubiquitin ligase complex that regulates NICD protein stability. In Drosophila, the gene most similar to sel-10, archipelago (ago), is required to destabilize Cyclin E proteins. However, available ago alleles do not affect the accumulation of NICD. The expression of a dominant-negative proteasome subunit stabilizes NICD in the Drosophila wing disc, and wild-type levels of proteasome activity are required for alternative cell fate decisions during sense organ development. In addition, treatment of cells with chemical proteasome inhibitors increases the accumulation of nuclear NICD. In this model, heph would regulate the mRNA for some element of the proteasome-dependent degradation of NICD (Dansereau. 2002 and references therein).

The most intriguing possibility is that heph may negatively regulate the translation of E(spl)-C mRNAs. The E(spl) complex bHLH genes are transcribed in response to Notch signaling and this is counteracted by inhibition of translation by the 3'-UTR's of E(spl)-C mRNAs. This inhibition is presumably mediated through the binding of factors to conserved sequences found in most E(spl)-C mRNAs as well as in genes of the Bearded family, another group of Notch mediators. In this model, loss of heph function would increase the stability of E(spl)-C mRNAs, resulting in amplification of the effects of transcriptional activation by Notch signaling. Increased expression of E(spl)-C members has been demonstrated to inhibit wing vein differentiation, although the ectopic expression of individual E(spl)-C members has not been demonstrated to induce ectopic wing margin. However, it is possible that the stabilization of multiple E(spl)-C mRNAs could result in more dramatic effects on the wing margin. Furthermore, E(spl)-C members have different transcription patterns and may have divergent roles downstream of Notch. If heph were to regulate a subset of the E(spl)-C mRNAs, it would explain the limited requirement of heph in various Notch-mediated signaling events (Dansereau. 2002).


GENE STRUCTURE

Genomic length - 140,000 bases

cDNA clone length - 3178 (alt1), 3491 (alt 2)

Bases in 5' UTR - 962 (alt1), 244 (alt2)

Exons - at least 15

Bases in 3' UTR - 509 (alt1), 877 (alt2)


PROTEIN STRUCTURE

Amino Acids - 568 (alt1), 608, 789 (alt2)

Structural Domains

Two full length Drosophila polypyrimidine tract binding protein (PTB) (Ptb) clones isolated from a stage 0-24 embryonic cDNA library were sequenced and found to encode a 581 residue protein that is highly conserved with the human and Caenorhabditis elegans orthologs of PTB, particularly in the RNA recognition motifs (RRM). The analyzed cDNAs did not contain evidence of alternative splicing within the protein coding domain and an examination of genomic sequence indicates that PTB is a single gene in Drosophila. This is in contrast with vertebrates, where PTB-related gene families and alternative splicing generate a number of PTB isoforms. The two Drosophila Ptb transcripts are distinguished, however, by unique 5' untranslated regions (UTRs) that are encoded by widely-separated sets of exons, and by differentially utilized poly-adenylation sites in the 3' UTR (Davis, 2002)

Heph is an RNA-binding protein with four RNA-recognition motifs (RRM1-4). Alternative splicing and three alternative start sites produce predicted protein variants: isoforms A and B include a bipartite nuclear localization signal (NLS). Only isoform A includes an N-terminal glutamine-rich domain (Q-rich). The Heph protein is highly related to the previously characterized RNA-binding proteins PTB/hnRNPI and Vg1RBP60 (Dansereau. 2002).


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

date revised: 8 December 2002

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