BIOLOGICAL OVERVIEW

In Drosophila oocytes, activation of Oskar translation from a transcript localized to the posterior pole is an essential step in the organization of the pole plasm, specialized cytoplasm that contains germline and abdominal body patterning determinants. Oskar is a component of polar granules, large particles associated with the pole plasm and the germline precursor pole cells of the embryo. aubergine mutants fail to translate oskar mRNA efficiently and are thus defective in posterior body patterning and pole cell formation. Aubergine protein is related to eukaryotic translation initiation factor 2C. In addition, Aubergine is recruited to the posterior pole in a vasa-dependent manner and is itself a polar granule component. Consistent with its presence in these structures, Aubergine is required for pole cell formation independently of its initial role in oskar translation. Unlike two other known polar granule components, Vasa and Oskar, Aubergine remains cytoplasmic after pole cell formation, suggesting that the roles of these proteins diverge during embryogenesis (Harris, 2001).

aubergine has also been implicated in regulation of post-transcriptional gene silencing in Drosophila. aubergine and spindle-E mutations cause a relief of Stellate and Su(Ste) silencing (Schmidt, 1999). Stellate derepression in the presence of the intact Su(Ste) locus has been observed as a result of aubergine and spindle-E (spn-E) mutations, also known as sting and homeless, respectively. The Aubergine protein has homologs involved in post-transcriptional gene silencing (PTGS) and double stranded RNA interference in plants, fungi, and animals. The spn-E gene encodes a putative RNA helicase that is also proposed as a participant in dsRNA-mediated silencing (Schmidt, 1999; Aravin, 2001).

Relief of Stellate< silencing occurs as a result of the spn-E¹ mutation. This was confirmed by studying the expression of the Ste-lacZ reporter construct in the spn-E¹/+ and spn-E¹/spn-E¹ males. The expression of ß-galactosidase in testes is greatly enhanced in spn-E¹/spn-E¹ males as compared to the heterozygous ones. The effects of the aub^sting-1 and spn-E¹ mutations on the level of sense and antisense Suppressor of Stellate [Su(Ste)] transcripts were assessed. Both mutations, when homozygous, have no effect on the level of antisense Su(Ste) transcripts, but increase the level of sense Su(Ste) RNA. Thus, a common mechanism, assisted by the Aubergine and Spindle-E proteins is operated in Su(Ste) dsRNA-mediated silencing of Stellate and sense Su(Ste) expression. The effect of the spn-E¹ mutation is restricted to the germline, since no increase in the level of sense Su(Ste) transcripts in the heads of homozygous flies was observed (Schmidt, 1999; Aravin, 2001).

Meiotic drive is defined as any alteration of meiosis or subsequent production of gametes that results in the biased transmission of a particular genotype. In other words, meiotic drive results in an excess of one type of gamete, X for example, over the production of the alternative gamete (Y in this case) instead of the usual ratio of 1:1 that is normally produced. Thus, meiotic drive is defined as any alteration in the meiotic process that distorts the normal 1:1 ratio of reciprocal gametes. Aubergine, in disguise as the mutation sting, is involved in crystal formation and meiotic drive in the male germ line of Drosophila. The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility correlates with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry, also known as Suppressor of Stellate) locus. In addition, their morphology is needle-like in Stellate (Ste) plus flies and star-shaped in Ste flies, once again as observed in cry males. The sting mutation leads to meiotic drive of the sex chromosome. The sting mutation results in an excess of X over Y sperm in the one hand and 0 over XY sperm on the other. The strength of the meiotic drive phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced (Schmidt, 1999).

Previous analysis of aub implicated it in two processes: the translational activation of a narrowly restricted subset of ovarian mRNAs, including osk and grk, and the subsequent localization of nos mRNA (Wilson, 1996). To investigate the molecular function of aub, the locus was positionally cloned. aub maps by meiotic recombination to genetic position 39 on the second chromosome. aub was subsequently mapped between P elements reported to be inserted in polytene regions 31E and 33A. RFLP mapping as well as clones and sequence information from the Berkeley Drosophila Genome Project were used to narrow the location of the aub gene to a small region at the boundary of polytene divisions 32C and 32D (Harris, 2001).

This region contains sting (Schmidt, 1999), a member of an ancient gene family that includes the gene for the eukaryotic translation initiation factor eIF2C (Zou, 1998). With support from three lines of evidence, aub was identified and it was concluded that aub and sting are the same gene. (1) In each of three aub mutant chromosomes, mutations predicted to truncate the Aub/Sting protein were identified. (2) Antibodies raised against a bacterially expressed C-terminal portion of Aub/Sting recognize a band of approximately 105 kDa in extracts from wild-type ovaries but not ovaries from aub mutants. A band corresponding to a similar molecular weight is produced by in vitro transcription and translation of an aub/sting cDNA. (3) Two genomic DNA fragments which include the aub/sting gene rescue aub mutants: aub^- mothers carrying a single copy of either transgene are fertile and produce viable offspring. Following tradition, the original name, aubergine, is used for this locus (Harris, 2001).

The Drosophila genome contains four other members of the eIF2C-like gene family. One of these is piwi, a close chromosomal neighbor of aub that acts in germline stem cell maintenance. Two additional members, Argonaute 2 and AGO1, are reported in the genome annotation. The latter is the closest known relative of eIF2C in flies and is presumably the Drosophila eIF2C homolog. The fifth family member, corresponding to the genomic sequence AE003107 and EST clot 2083, was identifed by tBLASTn searches of the BDGP databases using parts of Aub protein as the query sequence (Harris, 2001).

An early analysis of aub implicated it in two processes: the translational activation of a narrowly restricted subset of ovarian mRNAs, including osk and grk, and the subsequent localization of nos mRNA (Wilson, 1996). The latter function could be indirect, with Aub activating translation of a factor directly involved in nos mRNA localization, or Aub might itself play a more direct role in the process. The demonstration that Aub is related to the translation initiation factor eIF2C lends further support for a direct role of Aub in translational activation. Evidence that Aub is a component of polar granules and is required for pole cell formation adds an additional function and also suggests that its later role in localization of nos mRNA may be relatively direct (Harris, 2001).

Although the genetic pathways in which these eIF2C/piwi family members act have been identified, the direct activity through which any plays a role in development or RNAi has not been determined to date. The only gene product in the family for which a specific biochemical activity has been demonstrated is the translation initiation factor eIF2C (formerly Co-eIF-2A) (Zou, 1998). eIF2C purified from rabbit reticulocytes has two related activities that affect the ternary complex, which is composed of initiator methionine tRNA, GTP and eIF-2. The ternary complex binds the 40S ribosomal subunit to allow scanning for AUG codons in mRNA. Purified eIF2C stimulates formation of the ternary complex from components present at physiological levels, and it stabilizes the complex against dissociation in the presence of natural mRNAs (Roy, 1981; Bagchi, 1985; Chakravarty, 1985; Roy, 1988). The known activity of eIF2C offers a trivial explanation of the roles of other family members in multiple processes: each could simply enhance the translation of a protein required for that particular process. This seems unlikely, in large part because Piwi has been shown to be nuclear in both the germline and somatic cells in which its activity is required. Furthermore, Aub may play a direct role in nos mRNA localization. Nevertheless, the conserved domains that define this protein family should be expected to perform similar functions in the different proteins, and so some similarity to eIF2C function among other family members would not be surprising (Harris, 2001).

Aub protein may have biochemical activities similar to those of eIF2C. While eIF2C is predicted to play a global role in translation, mutants of aub only detectably affect the translation of a limited number of transcripts, revealing specificity in Aub function. Two broad classes of models are proposed for the role of Aub in message-specific translational activation. (1) Aub may simply elevate the level of eIF2C-like activity in the ovary, leading to enhanced translation of mRNAs that depend most on this initiation factor. Differential dependence would presumably reflect some feature of mRNA structure, such as folding or AUG sequence context, or association of the mRNA with other proteins or regulatory factors. A variation of this model invokes association of Aub with dependent mRNAs, either directly through an RNA-binding activity or indirectly via binding to other factors, effectively elevating the local concentration of the eIF2C-like activity. (2) Aub may perform an activity distinct from that of eIF2C but still be involved in some aspect of translation. As with the first model, the activity could be concentrated on dependent mRNAs through direct or indirect binding. A GFP-tagged form of Aub is not concentrated at the site of grk mRNA localization, and it has been argued that posterior concentration of Aub is not required for osk mRNA translation. Thus, if Aub does associate with dependent mRNAs as part of its role in translation, the interaction is likely to be transient (Harris, 2001).

Efforts to define the feature of transcripts that makes them dependent on Aub are constrained by the limited number of such mRNAs that have been identified: at present only osk and grk have been found. For osk mRNA, aub dependence is conferred in part by the 3' UTR (Wilson, 1996), a region known to bind multiple proteins and mediate both mRNA localization and translational repression. Therefore, Aub-dependence may be conferred by some aspect of translational repression or mRNA localization. The features of grk mRNA that confer aub-dependence are unknown (Harris, 2001).

osk mRNA is repressed by Bru and BicC, probably in concert with other factors. Bru also binds to grk, and this interaction has been suggested to mediate repression. If repression by Bru is indeed a shared feature of the osk and grk mRNAs, then Aub might inhibit or override this process. However, elimination of Bru-mediated repression by mutation of the BREs, the Bru-binding sites in the osk mRNA 3' UTR, does not abrogate the requirement for aub in translation of posteriorly localized osk mRNA (Wilson, 1996). Thus, Aub does not simply alleviate Bru-dependent repression at the posterior pole of the oocyte (although it may contribute to this process) (Harris, 2001).

One feature that is unquestionably shared by the osk and grk mRNAs is localization to specific domains within the oocyte. Perhaps tenacious binding of localization factors places unusual constraints on the translational apparatus that can only be overcome through the action of Aub. Supporting this possibility is the curious requirement for Aub in translation of posteriorly localized oskBRE^- mRNA, but not in the precocious translation that occurs prior to localization, when oskBRE^- mRNA is present throughout much of the oocyte (Wilson, 1996). Thus, posterior localization of osk mRNA correlates with the Aub-dependent phase of its translation. However, anterior localization of osk mRNA, achieved by exchanging the osk 3' UTR with the bcd mRNA localization signal, obviates the need for Aub. Therefore, mRNA localization in and of itself need not impose the requirement for Aub, although different localization signals may have unique properties that affect the relative translatability of their transcripts (Harris, 2001).

GFP-Aub colocalizes with Osk in polar granules in the pole plasm and pole cells. Moreover, since Osk and Vas are also largely colocalized in polar granules, it is inferred that the Aub-containing particles also include Vas. GFP-Aub is also highly enriched in the perinuclear zones of the nurse cells, a distribution shared by Vas and Tud, another polar granule component whose distribution was not monitored in these experiments. Notably, vas is required for the presence of normal levels of GFP-Aub in both the polar granules and the perinuclear zones. Pole plasm components have been suggested to be transported in particles from the nurse cells to the posterior ooplasm, and the results are consistent with such a scheme. The defect in GFP-Aub accumulation at the posterior in vas mutants may be a direct result of the initial failure to recruit Aub to the periphery of the nurse cell nuclei, a process that may well involve the assembly of complexes containing both Vas and Aub. In contrast, Stau is not required for the perinuclear localization of either Vas or GFP-Aub, and thus may only act later in the recruitment of particles to the posterior pole of the oocyte (Harris, 2001).

In the embryo, polar granules are present in the cytoplasm of pole cells, and nuclear bodies that are similar in appearance form in the nuclei of these cells. Evidence supporting a structural relationship between the two classes of particle has come from the demonstration that Vas is present in both. This work provides additional supporting evidence of such a relationship: further indication of structural similarity comes from the demonstration that Osk and Vas are colocalized in both nuclear and cytoplasmic aggregates, some of which have the peculiar donut-like shape that is characteristic of a subset of polar granules and nuclear bodies. However, the data also reveal that the cytoplasmic and nuclear particles are not identical in composition, since the nuclear bodies are distinguished from the polar granules by the absence of GFP-Aub. Nevertheless, Aub, like Osk and Vas, is involved in pole cell formation, and all are predominantly or exclusively cytoplasmic during that process. It is possible that later aspects of germ cell development require certain polar granule components, such as Osk or Vas, to act in the nucleus. Future experiments in which the selective partitioning of the various polar granule and nuclear body components is perturbed may provide insight into how pole cell fates are specified and maintained or lost during development (Harris, 2001).

sting mutation leads to a combination of a male sterile and maternal lethal effect in Drosophila melanogaster. In particular, the male sterility depends on a spectrum of meiotic abnormalities that closely resemble those produced by the deficiency of the Y-linked crystal locus. Cytological and genetic analyses show that the sti mutation induces the same three phenomena as in X/Y, cry^- males: (1) the formation of crystalline aggregates in primary spermatocytes; (2) meiotic nondisjunction of sex chromosomes and autosomes, and (3) meiotic drive of sex chromosomes and autosomes. As in the case of X/Y, cry^- males, the meiotic defects induced by the sti gene result from the activation of both the heterochromatic and euchromatic Ste sequence clusters, leading to the production of a protein that, depending on its amount, self-aggregates in needle- or star-shaped crystals (Schmidt, 1999).

The similarity of the meiotic effects to the situation in X/Y, cry^- males goes even further. In both cases, sex chromosome nondisjunction is correlated to Ste copy number, but this is not found with respect to drive. Thus, the Ste-sti and Ste-cry interactions are a system in which sex chromosome disjunction and drive are uncoupled, in contrast to the deficiencies of the X-Y pairing sites of the X heterochromatin that cause strongly correlated nondisjunction and meiotic drive (Schmidt, 1999).

The Northern analysis of the Ste expression in sti mutant flies led to the discovery that the Ste sequences are basically transcribed in several tissues of males and females that produce high-molecular-weight transcripts. The observations that the high-molecular-weight Ste transcripts are present in males carrying the W¹² X chromosome and lacking the euchromatic Ste cluster strongly suggest that these transcripts correspond to the heterochromatic Ste cluster. Unfortunately, at the present time, a viable X chromosome with a deletion of the heterochromatic Ste cluster is not available, it cannot be determined whether transcripts from the euchromatic cluster are also part of the high-molecular-weight fraction. Interestingly, a concomitant decrease of this fraction occurs with the production of the 750- to 850-nt Ste transcripts, which is more evident in the testes of cry^- males independent of the Ste allelic status or in the testes of cry⁺ homozygous sti males when they carry a Ste allele. This indicates that at least for the heterochromatic Ste sequences, the regulation of their expression in X/Y, cry^- testes could be post-transcriptional. It has already been shown that crystal can regulate the expression of Ste sequences at the transcriptional and splicing levels (Schmidt, 1999).

With the present results, it is not unreasonable to suggest that these two different levels of regulation reflect the fact that the heterochromatic and euchromatic clusters are under different regulatory mechanisms, namely that the euchromatic Ste sequences are under transcriptional control, while the heterochromatic sequences are mainly regulated at the post-transcriptional level (Schmidt, 1999).

The data clearly show that a gene has been identified that somehow interferes with the Ste-cry interaction system. Alternative hypotheses can be proposed to explain the interference of sti with the Ste-cry system. It is possible that both cry and sti are part of the same machinery that exerts control of Ste expression and, perhaps, additional genes in meiotic cells. However, it is possible that sti misfunction affects chromosome organization in meiotic cells, leading to the inactivation of the crystal locus, thus triggering Ste expression. In this case, there are at least two ways in which chromosomal alterations formally inactivate crystal. The sti mutation may induce Y chromosome loss or produce an alteration in the chromatin conformation of the crystal region. Careful analysis of homozygous sti male testes has revealed the presence of the Y loops in all mature spermatocytes, thus ruling out a sti-induced chromosome loss. Regarding the possible alteration in chromatin conformation, it is very difficult to perform a significant assay because the chromosomes are already altered as a result of the action of Ste protein. Therefore, at this time, there are no strong indications favoring one of the two alternative hypotheses (Schmidt, 1999).

The interaction between the autosomal sti and the X-chromosomal Ste gene opens the question of how these two partners have evolved. It is known, by Southern hybridizations, that the repetitive X-chromosomal Stellate gene exists only in D. melanogaster and its closest relatives, D. mauritiana and D. simulans (Livak, 1984). However, sequences homologous to at least parts of the Sti protein could be detected in a variety of organisms, ranging from the unicellular to higher plants and animals. Therefore, sti sequences should be detectable in more Drosophila species than those in which Ste sequences are found. This is indeed the case. The sting gene hybridizes even under stringent conditions with DNA from all species of the melanogaster subgroup, i.e., D. erecta, D. mauritiana, D. simulans, D. teissieri, and D. yacuba. Thus, the Ste-sti interaction is apparently a rather recent acquisition in some species of the melanogaster subgroup (Schmidt, 1999).

It is tempting to speculate that the primary sti function is related to the observed maternal lethality, and that this essential function is the one responsible for the conservation of Sti protein. Its function during spermatogenesis would then only be a secondary one. The modular structure of the upstream controlling regions favors this hypothesis; in these regions, the sequences necessary for expression in the ovary can clearly be separated from those that regulate expression in the male gonad. What, then, could be the primary function of the Sti protein? One possibility is that the wild-type function represses general or specific transcript processing. This is strongly supported by the Northern analysis of Ste expression, where processed Ste mRNA is found in mutant males. By this view, an induced out-of-phase gene expression should be responsible for the observed lethal maternal effect (Schmidt, 1999).

GENE STRUCTURE

cDNA clone length - 2761

Bases in 5' UTR - 28

Exons - 9

Bases in 3' UTR - 132

PROTEIN STRUCTURE

Amino Acids - 866

Structural Domains

The central and C-terminal portions of Aub contain two conserved regions, designated the PAZ and Piwi domains (Cerutti, 2000), which are encoded by a group of genes from organisms as diverse as plants, fungi and metazoans (including vertebrates). Recently, several of these genes have been characterized genetically and have been found to play essential roles in development. Both argonaute (ago1) and pinhead/zwille are required for maintenance of the axillary shoot meristem in Arabidopsis thaliana. In Drosophila, piwi has a demonstrated role in germline stem cell maintenance. Similarly, two Caenorhabditis elegans genes closely related to aub and piwi, prg-1 and prg-2, are also likely to be involved in germline proliferation. Other genes in the eIF2C/piwi family are implicated in mediating double-stranded RNA interference (RNAi) in C. elegans (rde-1) or the potentially related phenomena of post transcriptional gene silencing (PTGS) in Arabidopsis (ago1) and quelling in Neurospora (qde-2). The roles for ago1 in both PTGS and a cell fate decision reveal that a single gene in the family can carry out two functions, but it is not known if these functions are mechanistically distinct (Harris, 2001).

A databank search has revealed 28 significant matches in the nonredundant GenBank (probability for a chance match ranging from 6 x 10^-5 to 1 x 10^-115), as well as additional matches in the expressed sequence tag (EST) database. The vast majority of these sequences are derived from genome projects and therefore cannot provide any hint about a possible function of the Sti protein. The two exceptions are recently isolated genes from the plant Arabidopsis thaliana. The argonaute and zwille genes are involved in the regulation of the self-perpetuation of the shoot meristem. How this is achieved, however, is unknown, and the molecular function of sting-related proteins remains an enigma (Schmidt, 1999).

The most striking sequence similarity is localized in the C-terminal region of all the related proteins from the databank. This domain is homologous to the 82 amino acids close to the C terminus of Sti (amino acids 763–844). A multiple alignment and the consensus sequence of the hereafter-called 'sting domain' are presented. At least 13 different genes that code proteins with a sting domain have already been identified in the almost completely sequenced Caenorhabditis elegans genome. In less-advanced genome projects, three (Arabidopsis) and two (Drosophila) have been found to date. Although the totally sequenced Saccharomyces cerevisiae genome does not code any protein with a sting domain, the fission yeast Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum do code for at least one sting domain-containing protein, indicating that this domain is not unique to metazoa (Schmidt, 1999).

All but one of the putative C. elegans proteins found in the databank search contain blocks of sequence identities to the Sting protein outside the sting domain. These regions vary in size and are preferentially found in the C-terminal part of the proteins. Two proteins coded by genes D2030.6 and C01G5.2 display sequence identities throughout the Sti protein. The best alignment could be generated with the D2030.6 gene product. The high percentages of sequence identity (31.9%) and sequence similarity (52.1%) over the entire length of the proteins indicate that the product of gene D2030.6 could be the C. elegans ortholog of the Drosophila Sti protein. It is noteworthy that these two C. elegans proteins also have the smallest number of amino acid replacements within the sting domain (Schmidt, 1999).

date revised: 18 July 2002

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