BIOLOGICAL OVERVIEW

Apontic was identified in four different studies, each of which discovered different facets to the complex biological functions of this protein. Having first been identified as a modifier of Hox gene function in gnathal development, apontic may serve in a parallel pathway to Deformed and Sex combs reduced in the development of sclerite (cuticular) structures in the ventral portion of the gnathal region derived from maxillary and mandibular segments, with the exception of mouth hooks (Gellon, 1997). In the guise of tracheae defective, apontic has been shown shown to be required for the formation of the tracheal system during Drosophila embryogenesis. apt is not necessary for determining tracheal cell identity but for subsequent morphogenetic cell movements (Eulenberg, 1997). A third study involved a screen for enhancer trap lines that exhibit an early pattern of gene expression in cardiac precursor cells. apt proved to be adjacent to one plasmid insert showing expression in heart precursors, and apt mutant embryos show distinct abnormalities in heart morphology. A fourth study showed that Apt acts in concert with Bruno (also termed Arrest), a known translational repressor of Oskar, to repress the translation of OSK mRNA. Apt physically interacts with Bruno, and reducing the amount or activity of both Bru and Apt proteins appears to lead to a modest derepression of OSK translation (Lie, 1999).

While the observations made in the first three studies are more or less consistent in concluding that apontic might code for a transcription factor, the fourth study, showing that Apt binds to Bru and may regulate OSK mRNA translation rather convincingly leads to the conclusion that Apt is a cytoplasmic protein involved in translational control. This overview will deal with the fourth study and consider how these results impinge on the conclusions of the first three studies.

Proper control of OSK mRNA translation is essential and requires strict coordination with localization of the transcript to the posterior pole of the oocyte. Not surprisingly, translational regulation of OSK mRNA appears to be complex, and several factors involved in repression and activation have already been identified. The evidence for a direct role in OSK translation is strongest for Bru, a protein that binds specifically to regulatory sequences in the OSK mRNA 3' untranslated region (UTR). Another protein suggested to act on OSK in repression is p50, which was identified by virtue of its binding to OSK mRNA, but for which genetic confirmation of such a role has not been obtained. apt can be added to this roster of proteins and genes implicated in negative regulation of translation (Lie, 1999 and references).

It has been suggested that Apt functions as a transcription factor during embryogenesis, perhaps acting as a cofactor for certain Hox genes (Gellon, 1997 and Eulenberg, 1997). Two types of evidence have been presented to support this conclusion. (1) The Apt protein is highly concentrated in nuclei during most of embryogenesis, which strongly implicates a nuclear function. (2) The predicted structure of the Apt protein includes domains similar to those found in certain transcription factors. One is a short region enriched in glutamine residues, which may serve as a transcriptional activation domain. This by itself does not strongly support a role as a transcription factor, since similar glutamine-rich regions are found in a wide variety of Drosophila proteins, some of which are not involved in transcriptional regulation. The other domain is a potential bZIP motif. One part of this motif, the leucine zipper, is clearly present in Apt and may imply that the protein homodimerizes or forms a heterodimer with another protein in vivo. The second part of the bZIP motif, a flanking basic region, appears in an unusual form: certain amino acids known to be involved in DNA binding are present, but these are positioned much closer to the leucine zipper than in any other characterized bZIP domain. In addition, there are few basic amino acids. Consequently, Apt is either a rather unusual example of a bZIP transcription factor, or it may be a related protein whose function in the nucleus is less certain (Lie, 1999).

Apt is not always nuclear. Apt protein is persistently retained in the cytoplasm of early stage embryos even after other maternally provided proteins have shifted to the nuclei. This evidence for programmed control of the subcellular location of Apt suggests a requirement for Apt in the cytoplasm of early embryos. In the nurse cells of the ovary Apt protein is partitioned primarily to the cytoplasm. One possibility for how the subcellular distribution of Apt may be controlled is suggested by differences in APT mRNAs. The use of alternate 5' exons leads to variation at the amino terminus of the protein. Exon choice appears to vary during development, with one form of the mRNA found primarily among maternal transcripts while other forms are ubiquitous or most prevalent among zygotic transcripts. This pattern correlates well with the changing distribution of Apt protein: cytoplasmic Apt protein is synthesized largely or entirely from maternal mRNAs, while nuclear protein is synthesized from both maternal and zygotic mRNAs. Thus one form of the protein could be targeted to the nucleus and the other form to the cytoplasm (Lie, 1999).

Evidence implicating apt in the control of OSK translation is indirect. Biochemical experiments indicate that Bru and Apt proteins interact physically but provide no insight into the significance of the association. The genetic evidence -- head defects among progeny of mothers transheterozygous for apt and bruno (aret) mutations -- reveals a functionally significant interaction between the apt and aret genes but does not specify the exact nature of the interaction. Nevertheless, given the established role for Bru in repression of OSK mRNA translation, one likely explanation is that Bru and Apt both act in this process. Consequently, reducing the amount or activity of both Bru and Apt proteins could lead to a modest derepression of OSK translation. This interpretation is supported by the sensitivity of the phenotype to reduction of nanos gene dosage (Lie, 1999).

Although the genetic interaction of aret and apt supports a role for apt in repression of OSK mRNA translation, this function may not be essential. One of the apt mutants that shows a nanos-sensitive interaction with aret has only a modest phenotype in germline clonal analysis: a small fraction of the embryos from the homozygous mutant germlines display head defects. While this phenotype is consistent with a partial relaxation of the controls on OSK activity, it is inconsistent with a complete derepression of OSK mRNA translation. Could this particular mutant be a weak allele? This seems somewhat unlikely (but not impossible) since it displays a stronger genetic interaction with aret than does another allele, which has a strong arrested oogenesis phenotype in homozygous mutant germlines. Another possibility is that apt performs a redundant or partially redundant role in repression of OSK mRNA translation. An appealing feature of this model is that a candidate exists for a protein with overlapping function. Gunkel (1998) recently described a protein, p50, that also binds to the regions of the OSK mRNA bound by Apt; Apt and p50 could have similar roles in regulation of OSK expression. The gene encoding p50 has not been identified, so genetic tests of this model are not yet possible (Lie, 1999).

The demonstration that Apt is an RNA binding protein is somewhat unexpected, since none of the well-characterized RNA binding motifs appear in the predicted protein sequence. The ability of Apt to discriminate in its binding to certain regions of the OSK mRNA 3' UTR is striking, but its significance is uncertain, especially given the binding of Apt to a wide variety of other RNAs. In further characterization of apt function, it will be of interest to determine whether Apt RNA binding activity is important for proper regulation of OSK mRNA translation, or if the interaction of Apt and Bru proteins is sufficient. Notably, Apt protein does not colocalize with Bru and OSK mRNA to the posterior pole of the oocyte, raising the possibility that displacement of Apt from Bru may allow translational activation (Lie, 1999).

GENE STRUCTURE

The published sequences for apontic diverge in the 5' region, resulting in an additional six amino acids in the protein defined by the cDNA (Lie, 1999). This divergence can be simply explained by use of an alternative promoter and 5' exon, an interpretation that is consistent with the appearance in Northern blot analysis of a slightly smaller form of the mRNA during oogenesis and early embryogenesis. The ovarian cDNA described by Lie, 1999, contains 5' sequences provided by an exon located approximately 16 kB upstream of the 5' exon contained in the embryonic transcript (Lie, 1999 and Gellon et al., 1997).

Bases in 5' UTR - 249

Exons - 6

Bases in 3' UTR - 886

PROTEIN STRUCTURE

Amino Acids - 483

Structural Domains

The predicted structure of the Apt protein includes domains similar to those found in certain transcription factors. One is a short region enriched in glutamine residues, which may serve as a transcriptional activation domain. Apt also possesses a potential bZIP motif. One part of this motif, the leucine zipper, is clearly present in Apt and may imply that the protein homodimerizes or forms a heterodimer with another protein in vivo. The second part of the bZIP motif, a flanking basic region, appears in an unusual form: certain amino acids known to be involved in DNA binding are present, but these are positioned much closer to the leucine zipper than in any other characterized bZIP domain. In addition, there are few basic amino acids. Apt is also an RNA binding protein but does not possess any identifiable RNA-binding motif (Lie, 1999).

date revised: 15 April 99

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