InteractiveFly: GeneBrief

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

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.
BIOLOGICAL OVERVIEW

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).


REGULATION

Cellular internal ribosome entry segments: Structures, trans-acting factors and regulation of gene expression

Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. The nucleo-cytoplasmic shuttling protein PTB (hephaestus) (polypyrimidine tract-binding protein)/hnRNP I was identified as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 3' untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing (Besse, 2009).

The finding that exogenous RNAs fused to the oskar 3'UTR hitchhike on endogenous oskar molecules for their localization at the posterior pole of the oocyte revealed the capacity of oskar to oligomerize in vivo and assemble into high-order RNP particles containing multiple mRNA molecules (Hachet, 2004). Importantly, the oskar 3'UTR is not only sufficient, but also required for in vivo oligomerization, as exogenous RNAs harboring deletions in this region fail to hitchhike on endogenous oskar (Besse, 2009).

PTB was identified as a trans-acting factor required for formation of high-molecular-weight complexes in vitro, and for efficient copackaging of both 3'UTR-containing reporters and endogenous oskar mRNAs in vivo. This property correlates with the strong binding of PTB to multiple sites dispersed throughout the 3'UTR. Interestingly, a chaperone activity has been proposed for the vertebrate PTB, based on its capacity to bridge two separate regions of the FMDV IRES (Song, 2005), on the conformational changes in RNA induced upon its binding (Mitchell, 2003; Pickering, 2004), as well as on its role in remodeling of the Vg1 RNP complex (Lewis, 2008). This function is further supported by a structural analysis revealing that RRM3 and RRM4 of human PTB adopt a fixed and atypical orientation in which the RNA-binding surfaces of these domains are positioned away from each other (Oberstrass, 2005). As a consequence, RRM3 and RRM4 have the capacity to bring distantly located tracts into close proximity and thus, induce looping of the bound RNA. Given that the amino acid composition of these two RNA-binding domains and their linker region is highly conserved, Drosophila PTB likely folds and functions similarly to its mammalian counterpart. In the context of oskar mRNA, PTB binding may therefore induce specific RNA folding required to establish the RNA-RNA or RNA-protein interactions essential for multimerization of oskar mRNA. Alternatively, PTB may itself bridge different oskar RNA molecules and nucleate the assembly of multimolecular complexes (Besse, 2009).

Repression of oskar mRNA translation is a complex process involving both cap-dependent and cap-independent mechanisms, as well as the presence of 5? and 3' regulatory regions. The phenotype of oocytes with reduced PTB levels indicates that Drosophila PTB, while dispensable for the transport of oskar mRNA, is required for translational repression of the localizing mRNA. Mammalian PTB is already known to regulate translation, mainly by promoting cap-independent translation initiation. PTB binds the IRES located on the 5'UTR of cellular and viral RNAs, thus enhancing the recruitment of trans-acting factors and ribosomes (Stoneley, 2004; Jang, 2006; Semler, 2008). Recent reports indicate that PTB can also promote translation of specific mRNAs when bound to their 3'UTRs (Reyes, 2007; Galban, 2008). However, PTB does not exclusively act as a translational activator, as its interaction with the IRES present in unr and bip mRNAs has been shown to down-regulate their activity (Kim, 2000; Cornelis, 2005). The results of this study suggest that Drosophila PTB also acts as a translational repressor when bound to the 3'UTR of a target mRNA (Besse, 2009).

The mechanism by which PTB enhances or represses translation of target mRNAs remains elusive. The most accepted hypothesis, which may also explain the multiple roles of PTB in RNA regulation, is its capacity to act as a chaperone molecule, promoting the folding of RNAs into specific conformations, thereby modulating the binding of other regulatory proteins. Thus, depending on the specific structure adopted by the RNA, the cellular context, and the binding of other regulatory trans-acting factors, PTB may either promote or inhibit recruitment of the translation machinery. In the case of oskar mRNA, however, the results show that PTB is not required for the binding of the two best-characterized oskar translational repressors, namely, Bruno and Hrp48. Although the possibility that PTB influences the activity of these proteins cannot be excluded, PTB function seems beyond the simple recruitment of oskar main translation repressors (Besse, 2009).

Significantly, the results establish an in vivo link between oskar translation repression and multimerization of oskar 3'UTR-containing RNAs. An attractive possibility is that PTB promotes the formation of densely packed oskar RNP particles, thereby rendering the mRNA inaccessible to the translation machinery. A similar cap-independent function has been proposed for the translational repressor Bruno, based on its ability to promote in vitro oligomerization of a 3'UTR fragment containing duplicated Bruno response elements (BREs), together with its capacity to nucleate the assembly of heavy silencing particles. However, it was observed that BREs are neither strictly required nor sufficient for the hitchhiking of oskar 3'UTR-containing RNAs on endogenous oskar in vivo. Therefore, other trans-acting factors and cis-acting sequences likely contribute to the formation of high-order oskar RNPs in the oocyte. In this context, the chaperone activity of PTB may be essential to promote multimerization of oskar molecules, which would ultimately contribute to their complete translational repression. In contrast, the oskar phenotype of ptb mutant oocytes suggests that the assembly of high-order RNP complexes does not play a significant role in oskar mRNA posterior transport (Besse, 2009).

The assembly of RNP complexes competent for mRNA localization and precise translational control has been suggested to occur in a stepwise manner, some factors associating with the mRNA in the nucleus, and being essential for the subsequent recruitment of other components in the cell cytoplasm. For example, nucleolar association of the RNA-binding protein She2p with its mRNA target ash1 was recently proposed to be an essential step in the assembly of translationally silenced localizing ash1 RNP complexes in yeast. Some oskar translation repressors have been shown to localize both in the nucleus and in the cytoplasm of germ cells. However, whether these regulators are recruited to the oskar complex in the nucleus and whether nuclear association of these factors is required for subsequent translation silencing have not been tested so far (Besse, 2009).

PTB belongs to the hnRNP family of nucleo-cytoplasmic shuttling RNA-binding proteins, which regulate different aspects of RNA metabolism both in the nucleus and in the cytoplasm of eukaryotic cells. Consistent with this, it was observed that Drosophila PTB not only colocalizes with oskar in the oocyte cytoplasm, but also strongly accumulates in the nuclei of germ cells. Given that the nuclear association of PTB with Vg1 mRNA has been proposed to control the subsequent localization of these transcripts in the cytoplasm of Xenopus oocytes (Kress, 2004), this study tested whether the association of Drosophila PTB with oskar mRNA in the nuclei of germ cells is required for its translation repression activity. Notably, it was found that a cytoplasmic version of PTB localizes to the posterior pole of wild-type and ptb mutant oocytes and that this localization is oskar-dependent, strongly suggesting that it is still able to associate with oskar mRNA. More importantly, the mutant GFP-PTB-δNLS is competent in oskar translation repression. Although it is possible that endogenous PTB is loaded onto oskar RNP complexes in the nucleus of germ cells, these data suggest that nuclear recruitment of PTB is not a prerequisite for the formation of translationally silenced complexes. This analysis supports a model in which the complex behavior of RNP particles is controlled by the independent association of specific protein modules in different cell compartments. It also provides further evidence for the reorganization of RNP complexes upon translocation into the cytoplasm (Besse, 2009).


DEVELOPMENTAL BIOLOGY

Embryonic

Digoxygenin-labeled antisense RNA probes directed against the entire Ptb coding region were prepared and used for in situ hybridization assays to determine the pattern of Ptb expression during Drosophila embryogenesis. These assays show that the Ptb mRNA is supplied maternally and is distributed uniformly in the newly deposited embryo. At stage 3, Ptb message becomes restricted to domains fated to become embryonic mesoderm, although it is not yet clear whether this pattern is generated by new transcription or through the selective degradation of pre-existing mRNA. At stage 5, Ptb transcripts are restricted to the mesoderm, where by stage 10, expression is localized to dorsal patches that will later become lateral stripes by stage 13. The mesodermal pattern of Ptb expression is consistent with its function in muscle development, which is a role previously described for PTB in vertebrates. In stage 18 embryos, PTB expression in the mesoderm is lost and instead becomes patterned in the developing central nervous system (CNS), where it is strongly expressed in several distinct foci in the brain and in a subset of unidentified cells in the ventral nerve cord (Davis, 2002).

The embryonic expression of Ptb in the mesoderm and CNS is consistent with the function of PTB as a regulator of gene expression in vertebrate muscle and neuronal tissues. However, the tissue-specificity of expression in the Drosophila embryo is in contrast to the general expression of Ptb found in mammals and provides evidence that PTB functions in the specification of mesodermal and neuronal lineages during development (Davis, 2002).

Polypyrimidine tract binding protein (PTB) is a member of the hnRNP family of RNA binding proteins that functions in a number of processes important for the regulation of mRNA metabolism and gene expression. Specifically, PTB binds polypyrimidine-rich intronic elements upstream of alternatively spliced exons to antagonize the binding of the essential U2AF splicing factor and repress the use of the regulated exons in specific tissues. Additionally, PTB interacts with elements that mediate 3-prime end processing of nascent transcripts and is required for the expression of viral mRNAs that contain an internal ribosome binding site. Tissue-specific or alternatively spliced isoforms of PTB are thought to have different gene regulatory properties, but little is known about the function and activity of PTB isoforms during development. The expression of PTB during Drosophila embryogenesis has been studied using in situ hybridization assays. PTB expression is patterned in the early embryo and occurs in specific mesodermal and neuronal lineages as well as in the imaginal discs and adult germline. These data indicate that PTB regulates gene expression in specific tissue lineages during development (Davis, 2002).

Larval, pupal and adult

Developing adult tissues also express Ptb. In the wing disc, transcripts are expressed throughout the wing blade, with some enhanced expression in the presumptive wing margin. The early developing eye expresses Ptb in the morphogenetic furrow, although this expression decreases in later stage eye discs. PTB expression has also been reported in mammalian germline, particularly in the testis. In situ hybridization analyses of Drosophila also show Ptb expression in the testis and ovaries. Specifically, testis expression of Ptb is found in primary spermatocytes, indicating a role in the post-mitotic development of the male gametes. In support of this, is the observation that the male-sterile mutation, Hephaestus (Castrillon, 1993), maps to the Ptb locus. In the ovaries, Ptb is highly expressed in the nurse cells of stage 10 egg chambers, which is consistent with the observation that Ptb is a maternally contributed transcript (Davis, 2002).


EFFECTS OF MUTATION

As part of a genetic analysis of polytene region 100EF of chromosome 3, a lethal complementation group was identified that causes wing defects in genetic mosaic animals. Four lethal alleles in the complementation group (hephe1, hephe2, hephj11B9 and heph03429) cause autonomous loss of wing vein differentiation in clones and all but hephe1, which is temperature-sensitive and likely to be hypomorphic, induce ectopic margin in genetic mosaics. All four lethal alleles fail to complement the male sterility of the previously identified (Castrillon, 1993) P-element-induced male sterile mutation of hephaestus [ms(3)heph2] (Dansereau. 2002).

The four lethal alleles and the male sterile allele of heph map to a transcription unit that is predicted to encode at least three isoforms of a protein with four RNA recognition motifs (RRMs). The P-elements of ms(3)heph2, heph03429 and hephj11B9 are inserted in large introns. hephe2 is an EMS-induced deletion of several coding exons, including the coding region for RRM1, RRM2 and part of RRM3. The temperature-sensitive hephe1 mutation is a mis-sense mutation that changes a conserved glycine (G) residue to a glutamine (Q) residue in the first predicted RRM domain of Heph. The mapping of all five heph alleles to a single transcription unit indicates the lethality, ectopic margin, loss of vein differentiation and male sterile phenotypes are all due to loss of function in the same gene (Dansereau. 2002).

Loss of heph in genetic mosaics induces ectopic wing margin. Based on initial observations that heph mutant clones disrupte normal wing pattern formation, several genetic mosaic analyses were performed with heph mutations. Similar results are found for genetic mosaics of all the strong heph alleles including heph point and P-element insertion mutants and with Df(3R)G45, a small deficiency that deletes heph along with a second lethal complementation group, modulo. Clones of Df(3R)G45 and of strong heph alleles are smaller than wild-type clones in twin spot experiments, indicating a growth disadvantage or increased cell death in the clone. Using pixel dimensions as an estimate of clone size, heph clones induced during mid-second instar are about 65% of the corresponding twin size by late-third instar. When heph clones were given a growth advantage using the Minute technique, the clone sizes increased but even clones induced during the first instar never occupied more than a small fraction of the wing blade. In adult wings, cell polarity and cell size (trichome density) are not apparently affected by heph loss. Mintute+ heph clones can differentiate all wing blade structures normally with the exception of veins. Mutant clones induced in larval imaginal discs are associated with ectopic wing margin, loss of wing margin and loss of wing veins. Clones induced throughout larval development are associated with ectopic wing margin when situated within a short distance of the endogenous wing margin. The ectopic margin of heph genetic mosaics always conforms to the original compartment identity and resembles the adjacent endogenous margin. The autonomy of the ectopic margin was tested in experiments marking heph mitotic clones with the bristle marker yellow (y). Clones were also marked with pawn (pwn) and twin spot experiments were performed marking the clones and twins with forked (f) and bald (bld), which affect both bristles and trichomes. In all of these experiments, the ectopic bristles are derived almost entirely from heph+ cells immediately adjacent to the clone with the occasional ectopic bristle induced from within the heph mutant tissue. The non-autonomy of bristle induction is especially clear when the heph growth disadvantage is partially rescued by generating marked Minute+ heph clones in a Minute background. The ectopic margin is induced along the border of the marked Minute+ heph mutant tissue when that border is close to the normal margin. Outside of this domain, mutant cells do not induce ectopic wing margin. The ectopic margin is associated with small outgrowths of wing blade tissue in clones located near the junction of the wing margin and the AP boundary. Dorsal or ventral heph mutant clones that apparently intersect the normal margin are associated with wing margin nicks (Dansereau. 2002).

heph clones induce the wing margin molecular markers Wingless, Cut and Achaete. The wing margin nicks and growth disadvantage caused by heph mutations could result from disruption of general processes required for cell survival. However, the ectopic margin phenotype indicates that heph plays a regulatory role in wing margin pattern formation. In order to determine what processes heph is disrupting at the presumptive wing margin, the expression of wing margin molecular markers was examined in heph mosaic wing imaginal discs. In normal margin development, wingless is expressed in two or three rows of cells straddling the DV compartment boundary and diffuses to induce wing margin bristle fate in cells flanking the wg expression domain. Thus, the heph ectopic margin phenotype can be explained if heph mutant cells express wg ectopically. Using specific antibodies, the expression of wg and cut, a second D/V boundary marker, was examined in heph genetic mosaic wing discs. In agreement with the distribution of ectopic margin in adult wings, ectopic Wg and Ct were observed in those heph mutant cells located within a few cell diameters of the boundary stripe of Wg and Ct expression. Ectopic Wg or Ct expression was never seen in heph mutant tissue further away from the endogenous margin. Since the expression of both wg and cut at the boundary depends on high levels of Notch activation, these results suggest that heph mutant cells near the endogenous boundary are Notch activated. Induction of bristles of the anterior wing margin by Wg depends on downstream target genes such as the proneural gene achaete. Thus, there is a strong prediction that anterior heph mutant clones should induce anterior margin-promoting genes such as ac. As predicted from the adult phenotype, ectopic Ac expression was observed surrounding heph mutant clones near the DV boundary with some ectopic Ac expression in the clones. The association of ectopic wg, cut and ac expression with heph mutant tissue suggests that ectopic margin is induced around heph clones by the same mechanisms acting during normal development (Dansereau. 2002).

Ser and Dl expression are reduced in heph mutant tissue. The precise expression of Wg in DV boundary cells that is present by the late third instar evolves through interaction between the Notch and Wg signaling pathways. In mid-second instar wing discs, the Notch pathway is activated to high levels along the boundary between dorsal and ventral cells by Ser, which is expressed dorsally, and Dl, which is expressed predominantly ventrally. Expression of Dl and Ser is dynamic, and the initial DV asymmetry disappears as the two ligands become expressed under the control of Wg signaling. By mid third instar, a broad stripe of cells along the DV boundary express Ser, Dl and wg. During late third larval instar, this broad domain evolves into a narrow stripe of cells expressing wg but not Ser or Dl, flanked on either side by cells expressing Ser and Dl but not wg. Wg secreted by the boundary cells is required to maintain high levels of Dl and Ser expression in the flanking cells. The high levels of Dl and Ser in the flanking cells serve two roles: (1) they signal back to adjacent boundary cells to maintain the high levels of Notch activation required for wg and cut expression; (2) in the flanking cells, Dl and Ser autonomously inactivate Notch signaling, which restricts Notch-dependent expression of wg and cut to the boundary. High levels of Notch signaling in late third instar boundary cells activate the expression of cut, which encodes a homeodomain protein required to repress Ser and Dl expression in the boundary cell domain (Dansereau. 2002).

The ectopic wing margin phenotype and association of ectopic wg, cut and ac expression with heph mutant tissue suggests that heph mutant cells situated near the endogenous DV boundary are highly Notch-activated and thus behave like boundary cells and induce wing margin fate in adjacent flanking cells. The complex interdependent signaling network at the DV boundary offers several possible mechanisms that could lead to Notch activation and the ectopic margin phenotype. Ectopic boundary cell fate and ectopic margin are induced by clones of cells mutant for dishevelled (dsh), which are deficient for Wg signal transduction, or by clones mutant for both Dl and Ser. In both cases, clones of cells in the flanking domains lose Dl and Ser expression and Notch becomes activated through signaling from the adjacent wild-type Dl- and Ser-expressing cells. In heph mutant cells, the levels of both Dl and Ser are autonomously decreased independent of clone position within the wing disc. The decrease in Dl and Ser protein levels in heph clones is sufficient to account for the ectopic induction of wg and cut expression in cells flanking the margin where Dl and Ser normally repress Notch. This reduction of Dl and Ser could be the result of loss of Wg signal transduction or to loss of Dl and Ser expression. Finally, autonomous activation of Notch signaling could result in heph mutant cells in the flanking domains assuming a boundary fate (Dansereau. 2002).

Disruption of Wg signal transduction is not a likely explanation for the loss of Ser and Dl expression in heph mutant clones. Clones mutant for heph in the antenna and leg have no pattern phenotypes and heph mutations do not enhance the phenotype of 'dishevelled-weak', a genetic background that is highly sensitive to dose changes in Wg pathway signaling components. Furthermore, the Wg target gene acheate can be activated in heph mutant cells, and expression of the Wg target gene Distal-less is not affected in heph clones. These results suggest that clones lacking heph are able to transduce the wg signal and that the primary effect of heph is not on wg signaling (Dansereau. 2002).

Further support that Notch signaling and not wg signaling is disrupted in heph mutants comes from a genetic interaction observed between heph mutants and fringeD4 (fngD4). fngD4 is a gain-of-function allele of fringe, a gene encoding a Notch-modifying glycosyltransferase. A DV fringe expression boundary is required for maximal activation of Notch signaling and proper induction of the DV organizer. Ectopic transcription of fng+ in fngD4 results in decreased Notch activation and wg expression at the DV boundary, causing loss of the wing margin and much of the wing blade. A decrease in the dose of a wg pathway component would be predicted to enhance the fngD4 phenotype. However, flies heterozygous for both heph and fngD4 have considerably more wing margin and wing blade than do flies heterozygous for fngD4 alone. A similar suppression has been reported for flies heterozygous for fngD4 and activating `Abruptex' alleles of Notch (NAx). The Abruptex phenotype probably results from an inability to repress Notch activation, and like heph, NAx mitotic clones are associated with ectopic margin within a few cell diameters of the endogenous margin, and cause a cell-autonomous loss of vein differentiation (Dansereau. 2002).

The ectopic margin phenotype is probably caused either by loss of Ser and Dl expression or autonomous activation of the Notch pathway in heph mutant cells. One consequence of Notch activation is the cleavage of the full-length receptor, which releases the Notch intra-cellular domain (NICD), allowing it to translocate from the membrane to the nucleus. To determine if Notch is activated by loss of heph activity, the distribution of Notch immunoreactivity was examined in heph genetic mosaic wing discs. An increase in Notch immunoreactivity was found in heph mutant cells, regardless of their position within the imaginal disc. This increase is specific to an antibody that recognizes the intracellular domain of Notch (NICD) and is found in the cell body away from the apical surface of the cell. Since this effect is not observed with an antibody to the extracellular domain of Notch, and the apical levels of Notch are very similar in heph mutant and wild-type tissue, accumulation of the full-length Notch is not apparently increased in heph mutant cells. Consistent with this observation, comparable changes in NICD immunostaining are found along the DV boundary, where the Notch pathway is active and Notch target genes are expressed at high levels. As further evidence that the changes in NICD immunoreactivity represent Notch activation, it has been found that heph clones generated in Su(H) mutant discs do not alter the levels or localization of NICD. While it is not possible to conclude that the increased levels of NICD are localized to the nucleus, these results are consistent with an increase in Notch activation in heph mutant cells, and with the proposed role for Su(H) in transporting NICD to the nucleus. These data suggest heph acts in all wing disc cells to repress Notch pathway function. This is consistent with a report (Davis, 2002) that heph mRNA is present uniformly in imaginal discs (Dansereau. 2002).

The level of Notch is elevated and the level of Dl is decreased in heph mutant cells regardless of their position in the imaginal disc. This reciprocal relationship is typical of most tissues where Notch signaling is acting and is generally the result of interdependent signaling causing autonomous inhibition of Dl expression in Notch-activated cells. Dl itself is sometimes required to repress Notch activation autonomously so the observed decrease in Dl levels could be either a cause or an effect of increased Notch activation. In order to distinguish between these possibilities, Dl expression was examined in cells mutant for both heph and Su(H). In mature third instar discs, Dl is expressed ubiquitously at a low level, and in elevated levels at the DV margin, in the presumptive wing veins and in the proneural clusters of the thorax. Discs from third instar larvae mutant for strong alleles of Su(H) lack most of the wing pouch, because of the absence of Notch signaling along the DV boundary, but they retain the low ubiquitous expression of Dl. It was reasoned that if Dl expression were still reduced in heph cells in the absence of Su(H), then heph might act directly on Dl expression. However, no change was observed in the low levels of Dl expression in heph clones generated in Su(H) imaginal discs, indicating that the decrease in Dl expression in heph cells depends on Su(H). The implication of this result is that heph directly affects Notch activity and indirectly reduces ligand expression (Dansereau. 2002).

On balance, the effects of heph on wing margin formation suggest that heph represses Notch pathway activity. The heph loss-of-function phenotype in the wing veins also suggests that heph directly affects Notch signaling. Lateral inhibition involving Notch and Epidermal Growth Factor Receptor (EGFR) signaling is required to refine pro-vein territories in the wing blade. The position of veins is set by the expression of rhomboid (rho) in stripes of cells oriented perpendicular to the DV compartment boundary in the wing pouch. Rho facilitates signaling through Egfr and Egfr activation is required for the vein fate. Loss of Egfr activity is epistatic to the wide vein phenotype of Notch mutants, indicating that Egfr activation induces provein regions, and subsequently Notch functions to restrict vein fate by refining the domain of rho expression. Dl mutant clones that span a vein territory produce thicker veins than normal because Dl is required in the vein to activate Notch in adjacent lateral provein cells. By contrast, heph clones covering a vein territory cell autonomously fail to differentiate as vein. Only when dorsal and ventral clones coincide does a vein appear to be completely missing. This phenotype is consistent with ectopic Notch activation in heph clones since it resembles the effects of activating Notch by a variety of different genetic manipulations. Furthermore, despite the reduction of Dl expression in heph clones, the heph mutant clones have a wing vein phenotype opposite that of Dl mutant clones. This is strong support for the interpretation that the reduction of Dl expression in heph clones is a consequence rather than a cause of the Notch activated heph phenotype (Dansereau. 2002).

To determine the epistatic relationship between heph and Dl, the wing vein phenotypes of double mutant clones were compared with clones lacking only heph or Dl. Clones of cells mutant for both heph and Dl cause a thick vein phenotype that is indistinguishable from the effects of Dl mutant clones. These phenotypes indicate that heph is not required for specification of vein fate, i.e., heph is not directly required for rho expression or Egfr activity. Two interpretations are suggested. The parsimonious interpretation that heph acts to repress Delta contradicts the loss of Dl staining in heph mutant tissue, and the lack of requirement for Dl in specifying vein fate. Another interpretation is that Notch must be activated by Dl before heph is required. That is, heph may attenuate the Notch signaling pathway in cells where Notch has already been activated by Dl (Dansereau. 2002).

Hephaestus functions specifically in the male germline

The mammalian polypyrimidine-tract binding protein (PTB), which is a heterogeneous ribonucleoprotein, is ubiquitously expressed. Unexpectedly, in Drosophila, the abundant transcript of hephaestus, referred to as dmPTB in this publication, is present only in males (third instar larval, pupal and adult stages) and in adult flies is restricted to the germline. Most importantly, a signal from the somatic sex-determination pathway that is dependent on the male-specific isoform of the doublesex protein (DSXM) regulates PTB, providing evidence for the necessity of soma -- germline communication in the differentiation of the male germline. Analysis of a P-element insertion directly links PTB function with male fertility. Specifically, loss of Drosophila PTB affects spermatid differentiation, resulting in the accumulation of cysts with elongated spermatids without producing fully separated motile sperms. This male-specific expression of PTB is conserved in D. virilis. Thus, PTB appears to be a particularly potent downstream target of the sex-determination pathway in the male germline, since it can regulate multiple mRNAs (Robida, 2003).

To analyze PTB function in vivo and complement studies with the vertebrate PTB, the Drosophila PTB was studied. Unexpectedly, dmPTB is expressed in adult males but not females, as determined by Northern analysis using the full-length cDNA probe. Since prior studies have not suggested that PTB has a sex-specific function or regulation, it remained possible that the abundant band results from cross-hybridization via an RRM, a common highly conserved RNA-binding domain. To exclude this possibility, several probes were prepared corresponding to divergent portions of the gene such as the 5' and 3' untranslated regions (5' and 3' UTRs) and the variable linker region between RRMs (inter-RRM). Each of the probes shows an identical male-specific signal. Consistent with this finding, BLAST results show that there is only one sequence match to the dmPTB cDNA (P-value 6.7e–290) in the Drosophila genome. These results confirm that this abundant mRNA expressed in adult males but not females is a genuine dmPTB transcript (Robida, 2003).

Previously, a large-scale P-element insertion mutagenesis screen for male sterility identified the hephaestus2 (heph2) mutation (Castrillon, 1993), which was later mapped to the dmPTB locus by the Drosophila Genome Project. Other P-element insertions into the dmPTB locus are homozygous lethal (Dansereau, 2002). However, the molecular basis for the male sterility of the heph2 mutant was not studied. Since homozygosity for the heph2 allele causes sterility in male but not female flies (Castrillon, 1993), it was reasoned that this phenotype might be due to the absence of the abundant male-specific dmPTB transcript. To directly test this hypothesis, the expression of dmPTB was analyzed in heph2 flies. The dmPTB transcript was present in both wild-type and heph2 heterozygous males but absent in heph2 homozygous males. Thus, the heph2 P-element insertion disrupts the expression of the male-specific dmPTB transcript (Robida, 2003).

This study provides the first evidence that there is a major male-specific transcript of the Drosophila PTB that is regulated by the somatic sex-determination pathway. The sex-specific function of the abundant dmPTB transcript is restricted to the male germline. A direct molecular link is found between male fertility and PTB function, which offers a molecular basis for the male sterility of the heph2 mutant (Robida, 2003).

It is postulated that the somatic sex-determination pathway, in a DSXM-dependent manner, provides a signal for the proliferation and differentiation of male germ cells, leading to the expression of dmPTB in the male germline. Since tra and dsx are dispensable within the germline, their effect from the somatic tissue is inductive in nature. Accordingly, the DSXF isoform in the female soma or lack of the DSXM isoform in the male soma would fail to provide an appropriate signal for the development of the male germ cells. Thus, dmPTB expression is indirectly regulated by DSXM in the male germline (Robida, 2003).

There are several differences in the mechanism of sex determination between somatic cells and the female germline, e.g. the mechanism by which the X:A ratio is sensed is different between the two cell types. Furthermore, sexual differentiation is entirely cell autonomous in somatic cells but also requires a somatic inductive signal(s) in germ cells. It is emphasized that, unlike other male-specific transcripts that are either functional in somatic cells or dispensable for germline sex determination and spermatogenesis, dmPTB function is necessary in the germline for spermatogenesis. Thus, dmPTB provides evidence for the necessity of soma-germline communication in the differentiation of the male germline (Robida, 2003).

Several interesting aspects of dmPTB regulation, however, remain to be addressed. For example, relatively little is known about the molecular nature of somatic- or germline-specific activation signals for dmPTB expression. Also, whether the relevant germline-specific signal is repressed in the female germline or is activated only in the male germline cannot be distinguish. Finally, the promoter elements that confer male germline-specific expression remain unknown (Robida, 2003).

The male-germline-specific function of the abundant dmPTB transcript reported in this study directly links dmPTB function to male fertility. Specifically, dmPTB is expressed in primary spermatocytes and affects spermatid differentiation, resulting in the accumulation of cysts with elongated spermatids, but fully separated motile sperms are not observed. This phenotype is reminiscent of the defect seen in late male-sterile mutants such as the individualization-deficient Clathrin heavy chain (Chc) mutant, suggesting that dmPTB may control a component(s) of the cytoskeletal machinery. The expression pattern of dmPTB is consistent with the observation that the majority of transcription in germ cells is limited to the premeiotic stages, although protein synthesis and significant morphological changes occur during postmeiotic spermatid differentiation. Accordingly, the idea is favored that dmPTB is expressed early during spermatogenesis but affects either directly or indirectly the events that occur or manifest late during spermatid differentiation. It is emphasized that many male-sterile mutants are known to show secondary effects even though such mutations affect processes early during spermatogenesis. Thus, dmPTB in the male germline may control multiple targets or steps during spermatogenesis. Consistent with the known RNA-binding functions of the mammalian PTB, it could regulate the splicing, polyadenylation or translation of potential mRNAs that participate in spermatogenesis (Robida, 2003).

The male-germline-specific function of dmPTB is not necessarily inconsistent with the ubiquitous expression and multiple known targets of the vertebrate PTB. To reconcile these differences, the idea is favored that dmPTB performs an additional non-sex-specific function(s) vital for both sexes in Drosophila. (1) The male-sterile heph2 mutant also affects viability of both sexes. (2) Other mutations in the dmPTB locus are homozygous lethal. The most likely explanation for the different phenotypes of these mutations is that, whereas the heph2 mutation perturbs the male germline function but partially supports the vital function, the ema mutation compromises both functions. (3) Based on in situ hybridizations, dmPTB transcripts are expressed in several cell lineages, and minor transcripts are observed in females only upon longer exposure (Robida, 2003).

Given that the dmPTB locus is large (>135 kb) and that there is an indication of two distinct 5' UTRs (distal and proximal), the simplest interpretation for the two phenotypes is that the abundant male germline-specifc transcript reported in this study corresponds to an mRNA that contains the distal 5' UTR and is likely transcribed from an upstream promoter. Accordingly, the idea is favored that a downstream promoter(s) possibly contributes low abundance transcript(s) that are expressed non-sex-specifically in many cell lineages (Davis, 2002). This situation is reminiscent of two types of Sxl transcripts arising from a sex-specific establishment promoter (Pe) that is transiently active early during development (blastoderm stage) in females and from a non-sex-specific maintenance promoter (Pm) that is active in both sexes later during development. Although PTB transcripts are expressed in both male and female gonads in mice and worms, the possibility that the PTB protein is functional only in the male germline or regulates male-germline- specific mRNA(s) in the gonads of these organisms cannot be excluded (Robida, 2003).

Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and actin protein accumulation in Drosophila embryo

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions (Wesley, 2011).

Data presented in this report show that the loss of hephaestus (dmPTB) function affects the ventral and the dorso-lateral regions of Drosophila embryos very differently. In the ventral region, development of the CNS is suppressed and there was a discernible depletion in the level of the Notch protein. Suppression of the CNS development is consistent with the known role of hephaestus as a negative regulator of canonical Notch signaling during wing and bristle development in the larval and pupal stages. Excess canonical Notch signaling is well known to suppress neurogenesis in embryos. Depletion of the Notch protein in the ventral region that could be explained using data from mammalian systems showing that Nintra/NICD is turned over by a proteolysis process linked to the activity of the transcription factor Mastermind. Thus, excess canonical Notch signaling could result in Notch protein depletion if the rate of Nintra/NICD production and degradation is higher than Notch synthesis. If that were the case, it would suggest that the mechanism responsible for down regulating Notch activity targets not Nintra/NICD production or degradation but Notch synthesis. Combining the data from heph alleles and the Nnd1-dse allele, it appears that most of Notch mRNA transcribed following Notch activation is targeted for degradation by a mechanism that requires the Notch 3' UTR and the dse, a temperature sensitive allele of Notch that produces constitutive and high levels of endogenous Notch activities at the restrictive temperature. It is possible that the Hephaestus protein is part of the RNP complex that regulates this mechanism. In its absence, Notch protein synthesis continues instead of being suppressed. There is growing evidence that ligand-independent canonical Notch signaling in involved in development. It would be interesting to know if this signaling is also affected by the Hephaestus-based down-regulation mechanism. Thus, understanding how exactly hephaestus negatively regulates canonical Notch activity might provide insights into an important aspect of Notch pathway regulation that was hitherto obscure: down-regulation after activation of Notch by a ligand. As many human diseases are linked to gain of canonical Notch signaling, a better understanding of hephaestus and Notch 3' UTR and dse functions might lead to novel mechanistic insights into these diseases (Wesley, 2011).

The surprising finding in this study is the different response of the dorso-lateral regions of the embryo to the loss of hephaestus function or the loss of negative regulation of Notch mRNA 3' processing (due to the Nnd1-dse mutation). The simplest explanation is that Notch function is not required in these regions and de-repression of Notch protein synthesis results in the accumulation of Notch protein in these regions (as there is no signaling dependent depletion). This explanation, however, does not account for Pericardin accumulation, actin accumulation, or the block in dorsal closure. Pericardin level during cardiogenesis is well established to depend on Notch activity. These studies confirm that Pericardin is absent when Notch activity is eliminated (i.e., in Notch null embryos). Studies of others show that Notch activity is associated with higher actin level. Thus, it is very likely that Notch function is required in the dorso-lateral region of the embryo and this function is in excess in mutant heph and Nnd1-dse embryos. (Wesley, 2011).

As Nintra/NICD expression does not lead to actin or Pericardin accumulation in the dorso-lateral region, the simplest explanation is that Notch function in the dorso-lateral region is not completely based on Nintra/NICD activity in the nucleus. There is evidence for the existence of Notch activity independent of Nintra/NICD. For example, a Notch function independent of Presenilin (the enzyme that is required for the release of Nintra/NICD. A similar Notch activity might be functioning in the dorso-lateral regions of the Drosophila embryo. The data suggest that this non-canonical Notch activity might be situated at the cell surface or in the cytoplasm and is involved in regulating actin levels and cell fusion. This inference is consistent with the finding of others that Notch activity other than the one based on Nintra/NICD is associated with actin accumulation in wing discs (Wesley, 2011).

This non-canonical Notch activity could be the predominant Notch activity in the dorso-lateral regions of the embryos but it cannot be the only Notch activity. It is well known that Nintra/NICD and canonical Notch signaling is required for the peripheral nervous system (PNS) development from the dorso-lateral regions. These studies show that the PNS development is also suppressed in heph03429 and Nnd1-dse embryos, which raises the question of why no depletion is seen of Notch and actin proteins in the dorso-lateral regions as a consequence of constitutive Nintra/NICD and canonical Notch signaling. There are two possible explanations. One, a minority of cells are involved in the PNS development. Two, the canonical Notch signaling activity might precede the non-canonical Notch signaling activity and the latter determines the ultimate phenotype. Regardless of which explanation is correct, it is remarkable that cells in the ventral and the dorso-lateral regions respond so differently to the loss of hephaestus function or to the loss of negative regulation of Notch activity due to the Nnd1-dse mutation. At an earlier stage (stage 8 or 9), these cells were all the same, as they all adopt the default neuronal fate in Notch or Delta null embryos. At later stages, the ventral epidermal cells appear to diverge by blocking non-canonical Notch signaling altogether. It appears that this block is not an intrinsic lack of competence because actin and Notch enriched cells are occasionally observed in the ventral region. The block is specific to hephaestus or actin-related Notch activity as the ventral epidermal cells participate in other the actin-dependent processes, for example those involved in producing the denticle belts (Wesley, 2011).

The Notch pathway is long known to be involved in actin and adhesion processes in Drosophila. Interestingly, many processes that depend on Notch or hephaestus activity undergo cell fusion or block them (e.g., myoblast fusion). In this regard, Notch functions in myogenesis are quite instructive. During myogenesis, Nintra/NICD and canonical Notch signaling is required to restrict the number of myoblasts. Not as well known is the fact that Notch activity is also required subsequently for myoblast fusion and differentiation. A Notch activity at these stages is also reported to affect the differentiation of the neighboring epidermal cells and this activity is not based on Nintra/NICD. These reports have not been examined in depth so far because nothing is known about the non-canonical Notch signaling mechanism. The dorso-lateral regions of heph03429 and Nnd1-dse embryos represent an excellent model system for exploring non-canonical Notch mechanism with an unusual empirical power: all aspects of this mechanism in the dorso-lateral regions of the embryos can be compared with the canonical Notch signaling pathway mechanism in the ventral region of the same embryo (Wesley, 2011).

The process that is defective in the ventral region of heph03429 and Nnd1-dse embryos (neuronal cell fate specification) is known precisely, but nothing is known about the process that is defective in the dorso-lateral regions. These data contain two clues to the latter process. One clue is a contrast-enhanced image of wild type and heph03429 embryos probed with the actin antibody. A close examination of this image reveals that the strong actin signals in the heph03429 embryo are more or less amplified and expanded versions of the above-background actin signals in the wild type embryo. The second clue is in heph03429 and Nnd1-dse embryos probed with phalloidin. It appears that these embryos form enlarged versions of the cable-like actin structures that traverse almost the entire length of the dorso-lateral regions in the body of the wild type embryo. It is quite possible that clusters of cells in the dorso-lateral regions undergo partial or full fusion to form actin scaffolds that maintain epithelia integrity during remodeling and migration. Hypertrophy of these actin scaffolds could be the defect in the dorso-lateral regions of heph03429 and Nnd1-dse embryos. At this juncture, the mechanism by which actin protein level is altered in these mutant embryos is unknown (Wesley, 2011).

heph03429 and Nnd1-dse embryos appear to reveal a new level of developmental organization: broad zones that are competent or refractory to non-canonical Notch signaling activity. It is not known what factors or mechanisms determine these zones. A diverse array of mechanisms is known to regulate Notch activity at the protein level, such as glycosylation, trafficking, and proteolytic processing. It is possible that the ventral and the dorso-lateral regions differ in these mechanisms. Understanding the mechanism underlying the zonation of Notch activity in Drosophila embryos might also have practical implications since the Notch pathway is an important regulator of stem cell differentiation and cancer development. It might help in understanding variations within and among stem cell or cancer populations. It is possible that certain populations are composed of cells with potential for only the canonical Notch signaling while others include cells with potential for both canonical and non-canonical Notch signaling. Such differences in potentials might explain why some stem cells just proliferate while others differentiate or why some cancer cells are begin while others are metastatic (Wesley, 2011).


EVOLUTIONARY HOMOLOGS

Cloning and characterization of PTB

A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing (Garcia-Blanco, 1989).

Studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7. Factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage have been identified using an RNA mobility-shift assay. A protein that exhibits these sequence specific RNA binding properties has been purified to apparent homogeneity. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: (1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and (2) the intron regulatory element that is involved in the repression of exon 7. These results demonstrate that the sequence requirements for PTB binding are different from those previously reported and show that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites (Mulligan, 1992).

Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, the RNA binding properties have been analyzed of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. The primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2 (Perez, 1997).

Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, the binding has been characterized of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. A new 60-kDa tissue-specific protein has been identified that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins (Markovtsov, 2000).

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays (Liu, 2002).

Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3'-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. A fourth rodent PTB paralog (smPTB) has been identified and characterized that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localizes to the nucleus, binds to RNA, and is able to regulate alternative splicing. It is suggested that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events (Gooding, 2003).

PTB protein is essential for the integrity of the perinucleolar compartment

The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported in this study. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by alpha-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process (Wang, 2003).

PTB protein interactions

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remains intact and is N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggest differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provide evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociates from PTB and binds new partners, including the U1--70K and SR proteins and therefore may acquire new functions (Shav-Tal, 2001).

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induces very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor (Gromak, 2003).

PTB and RNA localization in Xenopus

Cytoplasmic localization of mRNA molecules is a powerful mechanism for generating cell polarity. In vertebrates, one paradigm is localization of Vg1 RNA within the Xenopus oocyte, a process directed by recognition of a localization element within the Vg1 3' UTR. Specific base changes within the localization element abolish both localization in vivo and binding in vitro by a single protein, VgRBP60. VgRBP60 is homologous to a human hnRNP protein, hnRNP I, and combined immunolocalization and in situ hybridization demonstrate striking colocalization of hnRNP I and Vg1 RNA within the vegetal cytoplasm of the Xenopus oocyte. These results implicate a novel role in cytoplasmic RNA transport for this family of nuclear RNA-binding proteins (Cote, 1999).

Effects of PTB depletion

Mutually exclusive use of exons IIIb or IIIc in FGF-R2 transcripts requires the silencing of exon IIIb. This repression is mediated by silencer elements upstream and downstream of the exon. Both silencers bind the polypyrimidine tract binding protein (PTB) and PTB binding sites within these elements are required for efficient silencing of exon IIIb. Recruitment of MS2-PTB fusion proteins upstream or downstream of exon IIIb causes repression of this exon. Depletion of endogenous PTB using RNAi increases exon IIIb inclusion in transcripts derived from minigenes and from the endogenous FGF-R2 gene. These data demonstrate that PTB is a negative regulator of exon definition in vivo (Wagner, 2002).

Deletion of PTB binding sites and knockdown of PTB both leads to an approximately 3-fold increase in exon IIIb inclusion, whereas deletion of a complete intronic control element leads to a 10-fold increase in exon inclusion. The most likely explanation for these results is that PTB collaborates with other unidentified factors, which bind to the 5' control element. The need for multiple factors to bind adjacent elements to integrate an alternative splicing outcome has been noted in several cases. HnRNP H, hnRNP F, KSRP, and nPTB have been found to bind the downstream splicing enhancer, which is required for inclusion of the N1 in c-src mRNAs in neural tissues. The tissue-specific inclusion of the alternative exon 5 of cardiac troponin-T appears to require the integrated activity of PTB and members of the msl family of factors. PTB associates with FBP and Sam68 on the intron upstream of the regulated exon 7 in rat ß-tropomyosin transcripts. The need to regulate a vast number of alternative splicing events has been solved by the integration of the activity of a limited number of factors that by combinatorial assortment can lead to very large number of functional states. An elegant binary switch provided by a single alternative splicing factor, as is the case for Sxl protein in D. melanogaster, may be reserved for crucial decisions, such as sex determination, which are made very early in development (Wagner, 2002).

It is clear that the factors that mediate silencing of exon IIIb via the upstream intronic splicing silencer and the intronic control element are present and active in both fibroblasts and epithelial cells. How then is exon IIIb included in FGF-R2 mRNAs in epithelial cells? It is likely that a cell type-specific factor or perhaps a combination of factors results in the specific derepression of exon IIIb. Exon IIIb activating factors are recruited via the intronic activating sequence 2 and the upstream activating element ISAR; these sites work in concert and form a secondary structure that is required for their function. Given that intronic activating sequence 2 is embedded within intronic element, it is reasonable to predict that the IAS2-ISAR structure will disrupt the silencing topology. Thus exon IIIb inclusion in epithelial cells is likely achieved by countering the repression mechanism instituted by PTB and other yet nidentified splicing repressors (Wagner, 2002).

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired (Knoch, 2004).

PTB transport and modulation of PTB activity

PTB functions as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism (Zhang, 1999).

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. The nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB (Xie, 2003).

PTB mediates exon inclusion

PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. The binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB is antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminates enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation (Lou, 1999).

PTB and the stabilization of mRNA

Stabilization of insulin mRNA in response to glucose is a significant component of insulin production, but the mechanisms governing this process are unknown. Insulin mRNA is a highly abundant messenger and the content of this mRNA is mainly controlled by changes in messenger stability. Specific binding of the polypyrimidine tract-binding protein to a pyrimidine-rich sequence located in the 3'-untranslated region (3'-UTR) of insulin mRNA has been demonstrated. This binding is increased in vitro by dithiothreitol and in vivo by glucose. Inhibition of polypyrimidine tract-binding protein binding to the pyrimidine-rich sequence by mutation of the core binding site results in a destabilization of a reporter gene mRNA. Thus, glucose-induced binding of polypyrimidine tract-binding protein to the 3'-UTR of insulin mRNA could be a necessary event in the control of insulin mRNA levels (Tillmar, 2002).

Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Alternative skipping of PTB exon 11 leads to an mRNA that is removed by nonsense-mediated decay; this pathway consumes at least 20% of the PTB mRNA in HeLa cells. Exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. These findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to nonsense-mediated decay (Wollerton, 2004).

PTB mediates repression of splicing

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. Conserved intronic elements flanking N1 have been identified that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. These upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds (Chan, 1997).

The smooth muscle (SM) and nonmuscle (NM) isoforms of alpha-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat alpha-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3' splice site, while the NM exon uses a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5' splice site, and this steric repression can be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts can be activated by (1) truncation of the transcript between the SM polypyrimidine tract and exon, (2) addition of competitor RNAs containing the 3' end of the actinin intron or regulatory sequences from alpha-tropomyosin (TM), and (3) depletion of the splicing extract by using biotinylated alpha-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB is the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlates with their ability to bind PTB. Furthermore, repression of alpha-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA can be specifically restored by the addition of recombinant PTB. Thus, alpha-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3' splice site and by PTB (Southby, 1999).

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for intronic splicing activator and repressor) which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. An intronic element upstream of exon IIIb has been identified that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms are equally capable of mediating this effect. These results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb (Carstens, 2000).

The role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon was investigated. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. It is concluded that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation (Chou, 2000).

Inclusion of cardiac troponin T (cTNT) exon 5 in embryonic muscle requires conserved flanking intronic elements (MSEs). ETR-3, a member of the CELF family, binds U/G motifs in two MSEs and directly activates exon inclusion in vitro. Binding and activation by ETR-3 are directly antagonized by polypyrimidine tract binding protein (PTB). Dominant-negative mutants have been used to demonstrate that endogenous CELF and PTB activities are required for MSE-dependent activation and repression in muscle and nonmuscle cells, respectively. Combined use of CELF and PTB dominant-negative mutants provides an in vivo demonstration that antagonistic splicing activities exist within the same cells. It is concluded that cell-specific regulation results from the dominance of one state among actively competing regulatory states, rather than modulation of a nonregulated default state (Charlet-B., 2002).

The Xenopus alphafast-tropomyosin gene contains at its 3' end a composite internal/3' terminal exon (exon 9A9') which is subjected to three different patterns of splicing according to the cell type. Exon 9A9' is included as a terminal exon in the myotome and as an internal exon in adult striated muscles whereas it is skipped in non-muscle cells. An in vivo model has been developed based on transient expression of minigenes encompassing the regulated exon 9A9' in Xenopus oocytes and embryos. The different alpha-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and valuable tools have been constituted to seek regulatory sequences involved in exon 9A9' usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9' in non-muscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9'. Using UV cross-linking and immuno-precipitation experiments it has been shown that XPTB interacts with these PTB binding sites. Finally, it is shown that depletion of endogenous XPTB in Xenopus embryos using a morpholino based translational inhibition strategy results in exon 9A9' inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9' and suggest that PTB could be a major actor in the repression of regulated 3' terminal exon (Hamon, 2004).

PTB and internal ribosome entry

Polypyrimidine tract binding protein 1 (PTB: Drosophila homolog Hephaestus) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr; a single-stranded RNA binding protein which contains five cold shock domains, Drosophila homolog CG7015) is prebound. The Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation (Mitchell, 2003).

The regulatory mechanisms controlling cell death are complex, and in addition to control of transcription, the expression of proteins that are involved in apoptosis is regulated by control of translation. Indeed, many mRNAs whose protein products are involved in apoptosis are translated by the alternative mechanism of internal ribosome entry. This process is mediated by a complex RNA structural element located in the 5' untranslated region (UTR) of the mRNA termed an internal ribosome entry segment (IRES). During apoptosis cap-dependent translation initiation is very much reduced, yet expression of certain key proteins required for this process is maintained by internal ribosome entry. Thus c-myc, DAP5, and XIAP IRESes function to maintain expression of these proteins following apoptosis. Apaf-1 translation is solely initiated by internal ribosome entry, but to date the only situation where a small increase in Apaf-1 IRES function has been observed is following genotoxic stress. Given the importance of Apaf-1 during brain development, it is possible that the Apaf-1 IRES is required for expression of this protein in the developing brain. In this regard the FGF-2 IRES has been shown to be active in adult brain while in developing embryos both the FGF-2 and c-myc IRESes are active. This suggests that certain IRES trans-acting factors (ITAFs) are not present in the fully differentiated cell types, and these and additional studies have demonstrated that the function of certain cellular IRESes varies considerably with cell type. Most cellular IRESes are inactive in vitro, again suggesting an absolute requirement for ITAFs that are not present in these systems. However, very few ITAFs have been identified for cellular IRESes although the auto-antigen La has been shown to interact with the XIAP IRES and hnRNPC has been shown to interact with the PDGF IRES. The Apaf-1 IRES requires both polypyrimidine tract binding protein (PTB; a protein that has a role in regulating splicing as well as aiding internal ribosome entry of certain viral IRESes and upstream of N-ras). PTB only binds to the Apaf-1 IRES RNA if unr is prebound suggesting that unr is required to attain the correct structural conformation of the Apaf-1 IRES (Mitchell, 2003).

PTB involvement in transcription

HMGA2 is an architectural nuclear factor that plays an important role in development and tumorigenesis, but mechanisms regulating its expression are largely unknown. The proximal promoters of the mouse and human genes coding for HMGA2 contain a conserved polypyrimidine/polypurine (ppyr/ppur) element which constitutes a multiple binding site for Sp1 and Sp3 transcription factors. This region can adopt a single-stranded DNA conformation, as demonstrated in vitro by S1 nuclease sensitivity on supercoiled plasmids, indicative of an intramolecular triple-helical H-DNA structure. Moreover, PTB (polypyrimidine tract binding protein), a member of the hnRNP family, binds the pyrimidine strand of Hmga2 as well as similar ppyr/ppur elements of the c-Ki-ras (R.Y) and c-myc P1 promoters. Transfection experiments indicate that non-B-DNA conformers of the ppyr/ppur tract of the Hmga2 promoter contribute to positive transcriptional activity. A transcriptional mechanism is proposed, one acting on the Hmga2 non-B-DNA structure and functioning through interconversion between double-stranded and single-stranded DNA. Such a mechanism seems to be adopted by an increasing number of genes, mainly growth-related (Rustighi, 2002).

PTB and translation of viral mRNA

Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for internal ribosome entry site). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This assay was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES (Hunt, 1999).

Bip is a chaperone protein that can also regulate the unfolded protein response of the cell. Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. As of yet, no trans-acting factor possibly involved in this process has been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibit Bip IRES-dependent translation. However, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These results suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs (Kim, 2000).

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). The requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) is completely attenuated by substituting its IRES with that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicates that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) does not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F map onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES is characterized by chemical and enzymatic footprinting. The data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES; their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs (Pilipenko, 2000).

The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, it has been shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for NES activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor (Li, 2002).


REFERENCES

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Biological Overview

date revised: 10 February 2012

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