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).
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).
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).
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).
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hephaestus:
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
date revised: 10 May 2004
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