InteractiveFly: GeneBrief

boule : Biological Overview | References

Gene name - boule

Synonyms -

Cytological map position - 66F5--6

Function - RNA-binding protein

Keywords - spermatogenesis

Symbol - bol

FlyBase ID: FBgn0011206

Genetic map position - 3-[27]

Classification - RRM motif protein

Cellular location - nuclear and cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

The Drosophila gene boule is a homolog of the human gene Deleted in Azoospermia (DAZ), which, when mutated, causes azoospermia (no sperm production) by blocking meiotic cell divisions. Deletions of portions of the human Y chromosome long arm occur in a fraction of men with azoospermia. These deletions define a Y chromosomal region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. The AZF region contains the DAZ gene, which is transcribed in the adult testis and encodes an RNA binding protein (Reizo, 1995). In fact, multiple copies of DAZ (>99% identical in DNA sequence) are clustered in the AZF region and there exists a functional DAZ homolog (DAZH) on human chromosome 3 (Saxena, 1996).

boule and DAZ encode closely related proteins that contain a predicted RNA-binding motif; both loci are expressed exclusively in the testis (Eberhart, 1996). boule expression is limited to males and the boule transcript is absent from flies lacking a germ line, indicating that expression is testis specific. The appearance of boule transcript during the life cycle coincides with the onset of testis development and spermatogenesis. Spermatocytes are formed in boule mutants, but fail to undergo meiotic divisions (Castrillon; 1993, Eberhart, 1996 and Santel, 1997). Comparison of the localization of Cyclin A in boule mutant and in wild-type germ cells supports the conclusion that the meiotic prophase is normal in boule mutants. Although the meiotic prophase appears wild type in boule mutant germ cells. subsequent stages are aberrant. Cyclin A, which is exclusively cytoplasmic in the extended premeiotic G2, and enters the nucleus at the transition between G2 and M phases only to be degraded rapidly after nuclear translocation, persists in boule mutants (Eberhart, 1996).

What then is the target of Boule during meiosis? An important clue lies in the similarity of boule and twine. Twine (Twe) is required for meiotic entry in males: spermatocytes lacking twine function fail at the G2/M transition and, as a result, carry out incomplete differentiation of tetraploid spermatids. This phenotype is also produced by conditional alleles encoding the cyclin-dependent kinase Cdc2 (Alphey, 1992; Courtot,1992 and Sigrist, 1995), as well as by mutations in two other genes, boule and pelota, that encode proteins predicted to interact with RNA. Twine/cdc25 is a phosphatase that activates cdc2 kinase activity during meiotic divisions. Like the zygotic protein String, Twine activates cell cycle progression by removing phosphate groups from cdc2, the cyclin dependent kinase that forms heterodimers with Cyclin A and Cyclin B. Confirming this notion, ectopic expression of String, which is known to activate the cdc2 kinase before mitosis, results in a partial rescue of meiotic divisions in twine mutant testis (Sigrist, 1995).

Boule is required for Twine protein expression. By assaying the progress of spermatogenesis in the twine partial loss of function background, boule is a candidate in vivo regulator of twine. Spermatocytes from males with partial loss of function for twine and with only a single wild-type copy of boule fail to enter meiosis. Moreover, these cells do not carry out the G2/M transition; unlike wild-type or twine mutant spermatocytes, these double mutant cells fail either to relocalize cyclin A from the cytoplasm to the nucleus or to form bipolar spindles (Maines, 1999).

To explore the mechanism by which boule acts as an enhancer of twe mutants, Twine expression was examined in genetic backgrounds deficient for boule, using a reporter construct. Expression of the Twine reporter protein is dramatically reduced in a boule mutant compared with wild type. This reduction occurs at the level of protein, and not RNA, accumulation, since the amounts of twine–lacZ and endogenous twine RNA present are not reduced but are instead increased in the absence of boule. Given that, in wild-type flies, Twine RNA accumulates well before the onset of meiosis, these data indicate that (1) Twine is translationally regulated and (2) efficient Twine translation requires Boule (Maines, 1999).

Support for this hypothesis is provided by a study of the patterns of Boule localization and Twine expression in developing spermatocytes. Boule protein translocates from the nucleus to the cytoplasm just before the first meiotic division, and is required in the cytoplasm for meiotic entry (Cheng, 1998). Boule relocalization is coincident with the first detectable expression of the Twine reporter, consistent with cytoplasmic Boule being essential in the translation of Twine protein (Maines, 1999).

Heterologous Twine expression rescues the boule meiotic-entry defect. If boule mutants fail in meiotic entry because of inadequate accumulation of Twine protein, heterologous expression of Twine should drive meiotic entry in a boule mutant. To test this prediction, Twine was expressed in a boule-independent manner by placing twine under the control of the spermatocyte-specific beta2-tubulin gene promoter and the 5' and 3' untranslated sequences of the beta2-tubulin gene. Expression of twine in this context is sufficient to restore fertility to twine mutant males (Maines, 1999).

Introduction of the beta2-twine construct into a boule mutant drives meiotic entry, with some cells completing at least one division in all individuals studied. Meiotic spindles, which are absent in boule mutant males lacking the beta2-twine transgene, are easily apparent in transformed lines. Although the observed rescue could result from Twine mRNA overexpression, this seems unlikely for two reasons: (1) amounts of endogenous Twine mRNA are already quite high in boule mutants; (2) the rescue is not dose dependent and the presence of either one or two copies of the -beta2-twine transgene has no deleterious effect in the wild type. Furthermore, the activity of the beta2-twine construct in a boule mutant does not reflect a general ability to drive meiotic entry. For example, although the male sterile phenotype produced by mutation of the pelota gene closely resembles that produced in twine or boule mutants, the beta2-twine construct does not restore meiotic entry in a pelota male. Thus, the beta2-twine transgene appears to specifically compensate for the defect in endogenous Twine translation in a boule mutant, thereby restoring meiotic entry (Maines, 1999).

Boule cannot be required to stabilize the Twine mRNA, because the Twine message is abundant in a genetic background lacking boule activity. Instead, Boule is likely to play a part in Twine translation. Given that Boule contains an RNA-recognition motif (RRM), it is thought that Boule could influence Twine expression through direct binding to the Twine mRNA. Twine is not, however, the sole target of Boule activity, since boule mutants show defects in spermatid differentiation that are absent in twine mutant males (Eberhart, 1996). Taken together, these data indicate that Boule may be required for both meiosis and spermatid differentiation, and may have a role in coordinating these two events (Maines, 1999).

The boule and twine gene products act downstream of a second set of genes required for meiotic entry. The products of these genes, which include spermatocyte arrest (sa) and meiosis I arrest (mia), are also required for the expression of Twine protein but not of twine mRNA (White-Cooper, 1998). Mutations in these genes result in a failure to accumulate Boule protein. This inability of sa and mia mutants to express Boule and therefore Twine, presumably contributes to their failure to initiate meiotic divisions (Maines, 1999).

Although proteins of the Dazl family are required for fertility in several organisms, little is known about their biochemical function. Given the similarities in sequence and in expression patterns among Boule and other Dazl-family members, and also in the phenotypes induced by mutation of these proteins, it is suggested that a role in translation or translational control will be a general property of such proteins. Moreover, given that inactivation of the murine Dazl protein results in a proliferation defect, it is possible that transcripts encoding Cdc25 proteins will prove to be a general target for Daz-related proteins (Maines, 1999 and references therein).


Transcriptional Regulation

In spermatogenesis, a major transition occurs as the mitotically amplifying population of spermatogonia cease mitosis and develop into primary spermatocytes. These primary spermatocytes become committed to undergoing the meiotic divisions, and then differentiating into spermatozoa. This change in cell behavior is associated with a dramatic switch in the transcript profile: some genes are downregulated and many are upregulated or switched on for the first time. The 'meiotic arrest' genes of Drosophila are crucial for regulating transcription in primary spermatocytes. The Drosophila always early (aly) gene is involved in this switch in spermatocyte transcriptional regulation. aly coordinately regulates meiotic cell cycle progression and terminal differentiation during male gametogenesis. aly is required for transcription of key G2-M cell cycle control genes and of spermatid differentiation genes, and for maintenance of normal chromatin structure in primary spermatocytes. Although entry into spermatid differentiation is independent of progression through the meiotic divisions, these processes are subject to coordinate control, mediated by the meiotic arrest class of genes, including aly, can, mia and sa. The meiotic arrest genes are essential for the transcription of many mRNAs involved in spermatid differentiation, and thus are required for spermatid differentiation. The meiotic arrest genes also control accumulation of proteins involved in the meiotic divisions, e.g. the cdc25 homolog Twine, and thus link differentiation to the cell cycle. The meiotic arrest genes of Drosophila have been split into two classes, based on the mechanism by which they control accumulation of Twine. The can class (including can, mia and sa) post-transcriptionally regulate Twine production. By contrast aly regulates transcription of twine. Two other meiotic regulators, cyclin B and boule, are also transcriptional targets of aly, but not can, mia or sa (Jiang, 2002 and references therein).

Targets of Activity

Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, the protein that specifically binds to the promoter of spermatid-differentiation gene Sperm-specific dynein intermediate chain (Sdic) was purified and identified as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G2/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line (Mikhaylova, 2006).

Although the general regulators of transcription in testes have been extensively characterized, the gene-specific transcription factors have long been elusive. To characterize such factors, attempts were made to identify the protein that binds to the conserved positive regulatory element b2UE1/b2UE2/TSE (see Nurminsky, 1998, for TSE consensus) that is necessary for activity of the β(2)tubulin promoter in Drosophila testes and is present in the promoter of the testes-specific gene Sdic. Modulo is required for transcription of a number of spermatid-differentiation genes, including β(2)tubulin and Sdic. Expression of Modulo itself in testes is positively regulated by the meiotic-arrest genes at the posttranscriptional level and requires the DAZ/DAZLA homologue Boule, the protein that also controls the G2/M meiotic transition through posttranscriptional regulation of Cdc25/Twine (Mikhaylova, 2006).

The promoter of the testes-specific gene Sdic contains the TSE motif that shows similarities to the conserved elements b2UE1 and b2UE2 found in other testes-specific promoters. An abundant TSE-binding protein was detected in protein extracts from Drosophila testes but not from gonadectomized males by using EMSA. Formation of the DNA–protein complex was completely inhibited by a 100-fold molar excess of the unlabeled TSE probe. At the same time, the presence of a 104-fold molar excess of the heterologous double-stranded oligonucleotide competitor 1 in all EMSA reactions did not inhibit formation of the DNA–protein complex, indicating that binding of the protein is sequence-specific. Further addition of the different oligonucleotide competitor 2 in 100-fold excess to the probe did not interfere with the complex formation. To corroborate this finding, five more different heterologous oligonucleotides were tested using the same conditions, and none of them impeded complex formation. Thus, a protein that specifically binds to the conserved TSE promoter motif is up-regulated in testes and may be involved in transcriptional regulation of Sdic (Mikhaylova, 2006).

To identify the TSE-binding factor, a multistep procedure was developed for its biochemical purification from whole adult flies Modulo is a broadly expressed protein that has been detected in ovaries, embryonic epidermis and mesoderm, larval imaginal discs, salivary glands, and brain and in cultured cells. Western blot analysis showed that the size of Modulo differs between testes and somatic tissues represented by the gonadectomized males. In testes, mobility of the protein is more consistent with the predicted 60.3-kDa size of the Modulo polypeptide; no signal was detected in testes of the mod07570 male sterile mutant, thus confirming the specificity of the assay. However, the apparent molecular mass of the Modulo variant expressed in somatic tissues is ~50 kDa, which corresponds with the size of the TSE-binding protein that was purified from the whole-fly extracts and identified as Modulo (Mikhaylova, 2006).

A analysis indicated that the 50-kDa protein is a truncated variant missing the N terminus of the full-size Modulo. All of the identified trypsin-generated peptides were located in the C-terminal portion of the molecule, whereas not a single peptide was identified near the N terminus. In addition, an unusual peptide flanked with the trypsin cleavage site at only one (the C-terminal) end was repeatedly identified with high confidence during the analysis. The N-terminal end of the peptide thus possibly represents the N terminus of the truncated 50-kDa Modulo variant, and its position is consistent with the size of the protein (predicted molecular mass, 46.2 kDa). In this case, the 50-kDa variant is missing the highly acidic N-terminal domain that is present in the full-size protein (Mikhaylova, 2006).

The 50-kDa somatic Modulo variant is capable of specific binding to the TSE motif. Thus, a specific variant of Modulo is expressed in testes, where it is capable of specific binding to the TSE-containing promoters. This full-size Modulo variant contains the N-terminal acidic domain, the structure of which is characteristic for the acidic activators that facilitate assembly of the core transcription machinery on the promoter and recruitment of chromatin-remodeling factors. Known acidic activators interact with the TFIID complex and facilitate interaction of TFIID with TFIIA and TFIIB. In testes, TFIID is represented by a specific variant encoded by the meiotic-arrest genes of the sa group. Modulo coimmunoprecipitates with the testes-specific TFIID subunit Sa and, thus, probably interacts with the testes-specific TFIID during transcriptional activation of testes-specific genes (Mikhaylova, 2006).

The suggested activity of Modulo as transcriptional activator in testes is not consistent with its role in somatic tissues, where it is involved in multiple vital activities that probably include chromatin-mediated transcriptional repression [based on the demonstrated Su(var) phenotype of the modulo mutants]. Structural differences between the Modulo variants may underlie this apparent discrepancy. The 50-kDa somatic Modulo variant is able to bind to DNA but is missing the N-terminal acidic domain and, thus, cannot establish the activating interactions observed in testes. Instead, the somatic Modulo variant may contribute to repression of testes-specific genes in somatic tissues (Mikhaylova, 2006).

The Modulo-binding element TSE is present in the promoter of the testes-specific gene Sdic that is up-regulated in primary spermatocytes. To regulate Sdic expression, Modulo has to be present at the same or earlier stage of spermatogenesis. Localization of the zone of up-regulation of Modulo in adult whole-mount testes using immunofluorescence showed that this zone, indeed, precedes and overlaps the zone of Sdic expression. To visualize Sdic expression, advantage was taken of the Sdic::GFP fusion transgene. Modulo is weakly expressed in early spermatogonia and stem cells located at the tip of the testis, but is up-regulated in late spermatogonia/early spermatocytes. Within the cells, Modulo is localized in both the nucleus and the cytoplasm. Specificity of the immunofluorescence staining was confirmed by the absence of appreciable signal in testes of the mod07570 and achi1 mutants that show severe down-regulation of Modulo; also, no staining was observed in wild-type testes when the primary antibody was omitted (Mikhaylova, 2006).

Thus, in spermatogenesis, up-regulation of Modulo precedes activation of its putative regulation target, Sdic. Attempts were made to see whether this pattern is also present for the general transcriptional regulators (the meiotic-arrest genes) and their regulation targets (the spermatid-differentiation genes). To dissect the temporal order of expression of these genes, transcript levels were quantitated in testes dissected from developing larvae using real-time RT-PCR. In Drosophila, spermatogenesis begins early in larval development, and the first wave of meiosis commences at the time of pupation. All of the five tested spermatid-differentiation genes, including Sdic, show drastic up-regulation in late third-instar larvae, i.e., during spermatocyte maturation before meiotic divisions. This finding is consistent with the observed pattern of up-regulation of the Sdic::GFP transgene in adult testes. However, up-regulation of the three studied meiotic-arrest genes precedes up-regulation of the spermatid-differentiation genes. Hence, Modulo is up-regulated in the male germ line in a manner similar to known general transcriptional regulators, before the major wave of transcriptional activation that includes spermatid-differentiation genes (Mikhaylova, 2006).

Although knockout modulo mutations result in lethality, a male sterile hypomorphic mutation mod07570 has been described; thus, mutation, caused by a transposon insertion, results in testes-specific modulo knockdown. To analyze the role of Modulo in transcriptional regulation, transcripts of a number of spermatogenesis-related genes were quantitated in testes of the mod07570 mutant and of the wild type, using real-time RT-PCR. The constitutive transcript of the ribosomal protein gene Rp49 was used as the cDNA template-loading reference. The ubiquitous transcripts RpL9 and Act5C and the broadly expressed spermatogenesis-related genes des and twe were not significantly affected by the mod07570 mutation (Mikhaylova, 2006).

Conversely, a number of genes with testes-biased expression were down-regulated to various extents in the mod07570 mutant testes. In particular, several meiotic-arrest genes (including aly, can, nht, and rye) were down-regulated 5- to 7-fold. Among the 13 other testes-biased genes examined, 7 showed moderate 2- to 5-fold down-regulation. However, four testes-biased genes showed >10-fold down-regulation, and two more genes were down-regulated 7- to 9-fold. Thus, there is a subset of testes-biased genes [including Sdic, Ssl, fzo, dhod, β, and dj] that are specifically affected by the Modulo deficiency (Mikhaylova, 2006).

Because the meiotic-arrest genes themselves are involved in transcriptional regulation in testes, some effects of the Modulo deficiency may be mediated by their down-regulation. Such effects should be similar to the effects caused by mutations in the meiotic-arrest genes themselves. This possibility was addressed by analyzing gene expression patterns in testes of the meiotic-arrest mutants achi1, sa1, and Taf12LKG00946 (rye), and by comparing them to the pattern of gene expression in the mod07570 mutant testes. Among the 13 testes-biased genes analyzed, four genes were affected differently by the meiotic arrest and the modulo mutations. The genes Mst98Ca, Pros28.1B, and CG10934 were very sensitive to mutations in meiotic-arrest genes but not in modulo and, conversely, the gene Ssl did not show striking sensitivity to the mutations in the meiotic-arrest genes sa and rye but was severely affected in the modulo mutant. Therefore, the effect of Modulo deficiency on transcription in testes cannot be reduced to down-regulation of the meiotic-arrest genes. It is possible that such down-regulation leads to the subpar performance of the meiotic-arrest genes that is still sufficient to carry the germ line of the mod07570 mutant through the meiotic divisions but results in moderate (e.g., 3- to 4-fold) down-regulation of testes-biased genes, such as Mst98Ca, Pros28.1B, and CG10934. However, a more severe effect of the Modulo deficiency on a subset of spermatid-differentiation genes probably reflects disruption of gene-specific transcriptional regulation and provides a molecular basis for the spermatid-differentiation failure observed in the mod07570 mutant (Mikhaylova, 2006).

The subset of genes strongly affected in the mod07570 mutant includes Sdic and β(2)Tubulin. These genes possess the TSE-like Modulo-binding motifs and, thus, probably represent the direct regulatory targets of Modulo. Interestingly, it was not possible to detect specific binding of Modulo to the promoters of fzo and dj that are also strongly affected by the modulo mutation. At the same time, the studied dj promoter fragment contained all sequences necessary for efficient testes-specific transcription. This finding implies that Modulo has indirect target genes such as dj and, probably, fzo that may be regulated by transcription factors that are, in turn, under the control of Modulo. A broad survey of 96 transcriptional regulators expressed in testes identified nine putative transcription factors that are down-regulated >10-fold in the mod07570 mutant testes. Thus, mutation in modulo can lead to disruption of the downstream cascade of transcriptional regulation that includes Modulo-dependent transcription factors and their regulation targets (Mikhaylova, 2006).

To determine whether Modulo is sufficient to induce ectopic transcription of spermatid-differentiation genes, recombinant full-size Modulo was expressed in the Schneider-2 cultured cells under the control of metallothionein promoter. Stable transfected clones were selected, and expression of the transgene was induced by various concentrations of Cu2+ in the culture media. Unexpectedly, it was observed that the Schneider-2 cells naturally express the full-size Modulo variant. Nevertheless, these cells do not show significant expression of the Modulo-dependent testes-specific genes Sdic, Ssl, and dhod, and increase of the Modulo dose by induced expression of the transgene did not affect the levels of these transcripts. Therefore, other, presumably testes-specific factors, (such as the testes-specific TFIID) have to cooperate with Modulo to induce expression of spermatid-differentiation genes, thus defining tissue specificity of the Modulo-mediated transcriptional regulation (Mikhaylova, 2006).

To analyze the regulation of Modulo expression in testes, a number of mutants that control different stages of spermatogenesis were analyzed. In testes of the bam mutant, both the Modulo protein and modulo transcript are severely down-regulated. Thus, high levels of Modulo expression in the testes require the onset of the meiosis/differentiation program. Furthermore, mutations in the meiotic-arrest genes achi/vis, sa, and rye result in severe down-regulation of Modulo protein in testes; however, modulo transcription is not affected. Therefore, Modulo expression in the testes is regulated by the meiotic-arrest genes at posttranscriptonal levels, similar to the regulation of the meiotic entry control protein Cdc25/Twine. Translation of Twine in the testes requires the RNA-binding protein Boule. To investigate whether a similar mechanism is involved in the regulation of Modulo, testes of the boule mutants were examined, and it was found that Modulo expression in testes is severely affected by the Boule deficiency (Mikhaylova, 2006).

Regulation of Modulo expression in testes by Boule provides a mechanistic link between meiosis and spermatid differentiation in the male germ line. The meiotic-arrest genes are required for expression of a number of spermatogenesis-related genes, including boule. Boule is required for expression of Modulo, which, in turn, is necessary to maintain expression of several meiotic-arrest genes. These events establish a positive regulatory loop that sustains high levels of expression of Boule, Modulo, and the meiotic-arrest genes after the onset of the meiosis/differentiation program in spermatocytes. Boule further regulates the G2/M transition in meiosis by positive translational regulation of Cdc25/Twine, and Modulo and the products of the meiotic-arrest genes are required for expression of a number of spermatid-differentiation genes. Thus, the pathways that lead to meiosis and to expression of the spermatid-differentiation genes in the male germ line are integrated in a single mechanism to ensure coordinated execution of meiotic divisions and spermatid differentiation (Mikhaylova, 2006).

Regulation of cap-dependent translation by eIF4E inhibitory proteins

During spermatogenesis, cells coordinate differentiation with the meiotic cell cycle to generate functional gametes. The gene off-schedule (ofs) was identified as being essential for this coordinated control. During the meiotic G2 phase, Drosophila ofs mutant germ cells do not reach their proper size and fail to execute meiosis or significant differentiation. The accumulation of four cell cycle regulators -- Cyclin A, Boule, Twine and Roughex -- is altered in these mutants, indicating that ofs reveals a novel branch of the pathway controlling meiosis and differentiation. Ofs is homologous to eukaryotic translation initiation factor eIF4G. The level of ofs expression in spermatocytes is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G), suggesting that Ofs substitutes for this protein. Consistent with this, assays for association with mRNA cap complexes, as well as RNA-interference and phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based on these studies, it is speculated that spermatocytes monitor G2 growth as one means to coordinate the initiation of meiotic division and differentiation (Franklin-Dumont, 2007). A second studied, co-published with the Franklin-Dumont paper, has reported similar findings (Baker, 2007).

Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 5' cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 5' untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA's 5' end with the 40S ribosomal subunit. This ribosomal subunit comes 'pre-charged' as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 5' to 3' direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins (Richter, 2005).

The eIF4E-eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small -- about 15 amino-acid residues (Mader, 1995). Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. A clear demonstration of why the competition between eIF4G and other proteins for interaction with eIF4E is so effective in preventing translation comes from X-ray crystallographic analysis. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin; see Drosophila Thor) form nearly identical α-helical structures that lie along the same convex region of eIF4E, some distance from this protein's cap binding site (Marcotrigiano, 1997; Matsuo, 1997). Peptides with the general sequence YXXXXLphi, where phi is any hydrophobic amino acid, would probably form similar α-helical structures, implying that other proteins containing this peptide motif could control translation initiation (Richter, 2005).

The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E (Gingras, 1999). eIF4E-binding proteins interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins (Richter, 2005).

In spermatogenesis, progenitor cells must execute the meiotic divisions in coordination with acquiring the specialized morphology and functionality of sperm. This conserved process is particularly amenable to analysis in Drosophila. The fly testis is a blind-ended tube organized as an assembly line for spermatogenesis. Germline stem cells at the blind end give rise to gonialblasts, which divide mitotically four times with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonia. These cells exit the mitotic cycle and enter meiosis as spermatocytes, exhibiting an extended G2 phase characterized by a significant increase in cell mass and robust transcription. At the end of G2, the spermatocytes undergo the meiotic divisions and begin the conversion from round spermatids to specialized spermatozoa (Franklin-Dumont, 2007).

Ten 'spermatocyte arrest' genes are required for both meiosis and differentiation and are sorted into two classes according to their molecular targets and specific role in promoting transcription. The always early (aly) class affects the transcription of meiotic genes such boule, twine and cyclin B, as well as that of differentiation genes such as fuzzy onions (fzo) and don juan. Notably, these mutations do not effect transcription of other spermatocyte genes, such as pelota, cyclin A and roughex. The Aly class proteins are thought to alter chromatin structure to permit the high levels of transcription necessary in spermatocytes. The cannonball (can) class affects boule and twine expression post-transcriptionally only and has no effect on cyclin B. The post-transcriptional effects must be indirect, because all can class loci encode testis-specific components of the general transcriptional machinery. Together, the spermatocyte arrest genes reveal how a diverse set of genes is selectively transcribed in spermatocytes (Franklin-Dumont, 2007).

The transcriptional regulatory pathway does not address the timing of meiotic entry and differentiation, however. Although transcripts necessary for these processes accumulate in early spermatocytes, the corresponding proteins do not appear until much later. Because there is little, if any, transcription after the G2-M transition in flies, spermatocytes must delay meiotic division until all the necessary transcripts have accumulated. A similar dilemma exists during the mitotic cycle in yeast. For cells to maintain the same average size over several divisions, control points act during the gap phases and allow cell cycle progression only when the cell has reached a threshold size, with G1 predominating in budding yeast and G2 in fission yeast. Cell growth rates also feed back on mitotic cell cycle progression in Drosophila cells. Less is known about how growth might affect the specialized meiotic cell cycle (Franklin-Dumont, 2007).

Identification and characterization of off-schedule provides evidence that cell growth is linked to the coordination of meiosis and differentiation. Spermatocytes in ofs mutant males fail to execute the G2-M transition of meiosis or substantive post-meiotic differentiation and have a significant cell size defect. The Off-schedule protein resembles the eukaryotic initiation factor 4G (eIF4G), which is a member of the eIF4F translation initiation complex and bridges mature mRNAs and the ribosome (Prevot, 2003). The eIF4G activity of Ofs is apparent in its ability to associate with mRNA caps and to functionally replace canonical eIF4G in cell culture. Because translation is primarily regulated at initiation, eIF4G is instrumental in determining the translational capacity of a cell and thus its ability to accumulate mass. Thus, the ofs mutant phenotype suggests that sufficient cell mass must accumulate before spermatocytes execute meiosis and differentiation (Franklin-Dumont, 2007).

Alignment among eIF4G sequences suggests that Ofs would be part of the eIF4F complex with eIF4A and eIF4E, and demonstration of its association with 7-methyl GTP Sepharose strongly supports this. Although binding of Ofs directly to eIF4A was not measured, alignment of human and fly eIF4G proteins shows conservation of three out of four sets of amino acids necessary to bind eIF4A (Imataka, 1997). Of 12 crucial residues, ten were identical in Ofs, one was a conservative (L>I) change, and the twelfth diverged in Drosophila eIF4G as well. With regard to eIF4E binding, the putative binding site in Ofs has an arginine substituted for the usual hydrophobic residue. However, a similar substitution is tolerated in Drosophila eIF4E binding protein 1 (Miron, 2001), and Baker (2007) presents evidence for interaction with Drosophila eIF4E1. Taken together, it is quite likely that Ofs participates in cap-dependent translation initiation (Franklin-Dumont, 2007).

eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins encoded in the fly genome. One other candidate, l(2)01424, is more related to the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G proteins (Takahashi, 2005). Although the novel N-terminus of Ofs raised the possibility that it would play a role distinct from eIF4G, the data suggest that Ofs can act as the only eIF4G in cultured cells. Whether these two proteins always act redundantly in vivo cannot be assessed without mutations in eIF4G. Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of a special class of mRNAs, specific to spermatocyte development. Perhaps sequences in its novel N-terminus assist in such a role. Although further experiments are needed to distinguish between these possibilities, one reason for a distinction between spermatocytes and other cells might be in their respective mode of growth control. In cultured eIF4G-deficient mitotic cells, the cell cycle effect observed was on G1, whereas the defect in spermatocyte progression is in G2. Although the G1-S transition is the major control point for growth sensing in mitotic cells of the fly, G2 might make more sense as the control point for meiosis, because it is during this phase of the cycle that spermatocytes need to prepare not just for division, but for differentiation. Furthermore, spermatocytes might commit to the meiotic cycle, versus returning to the mitotic cycle, during G2, as is the case for the yeast Saccharomyces cerevisiae. Perhaps expressing a unique eIF4G (Ofs) in spermatocytes helps serve this role. Given the functional role for ofs, it is proposed that ofs henceforth be known as eIF4G2 (Franklin-Dumont, 2007).

Because ofs (eIF4G2) encodes the predominant eIF4G in spermatocytes, one might expect that mutant cells would exhibit decreased translation of many mRNAs. Just as a striking delay was found in in Boule accumulation, other proteins would be expected to be similarly affected. Such a global deficit could account for the delayed development of these cells, and would be predicted to influence cell size, because the translational capacity of a cell predicts its ability to accumulate mass. Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was their small size. Yet, Aly accumulation appeared normal and Rux protein appeared to accumulate to an excess degree in early spermatocytes. These data demonstrate that some mRNAs are not affected by the translational deficit, and raise an alternative scenario wherein spermatocytes actively monitor their size. If they do not achieve proper growth, a checkpoint is induced to prohibit meiosis and differentiation. Because meiosis involves two cell divisions with little intervening interphase, size monitoring would be especially important before these cells commit to divide (Franklin-Dumont, 2007).

Circumstantial support for a growth checkpoint includes the accumulation of the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this model, the postulated checkpoint causes the striking delay in the accumulation of Boule, which, in turn, explains the delay in Twine accumulation. Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak through the checkpoint, just as eventually occurs in mitotic checkpoints. However, by then, Cyclin A has been degraded, and without it, the eventual accumulation of Twine cannot trigger meiosis, so the checkpoint has succeeded (Franklin-Dumont, 2007).

To establish that a checkpoint exists, one would need to identify the sensor, which detects the problem, and effectors, which execute inhibitory functions until the cell resolves the problem. No candidate is available for the sensor that detects growth at this time, nor for effectors controlling differentiation. However, it can be speculated that Rux is one effector regulating the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2 kinase activity (Avedisov, 2000). Rux is not the only effector regulating meiosis, however. Previous work showed that directly increasing the level of Rux only blocked entry into the second meiotic division (Gönczy, 1994). Consequently, the accumulation of Rux that is observed in eIF4G2 mutants cannot fully explain the absence of the first meiotic division or the defect in differentiation. As would be typical for cell cycle regulation, several effectors must be activated at once to completely block the G2-M transition (Franklin-Dumont, 2007).

The existence of other effectors could explain why forcing early Twine accumulation failed to restore meiotic entry to eIF4G2 mutants in a rux background. Alternatively, there might be additional positive factors necessary for G2-M transition that have not accumulated in eIF4G2 spermatocytes. Consistent with this, prior work driving expression of another Cdc25, string (stg), in early spermatocytes directed a normal rather than a precocious G2-M transition. Thus, advancing Cdc25 activity is insufficient to trigger a precocious G2-M even in the absence of a growth defect. Perhaps early spermatocytes have not had enough time to accumulate an essential component, such as Cyclin B, for the meiotic divisions. It was found that eIF4G2 mutant clones exhibit Cyclin B levels comparable to neighboring heterozygous cells. However, there is a peak in Cyclin B accumulation just prior to meiosis I, and Baker (2007) describes a deficit of this Cyclin B peak in eIF4G2 mutants. Thus, Cyclin B remains a candidate factor (Franklin-Dumont, 2007).

Whether a growth checkpoint exists or not, mass accumulation could be used to time the G2-M transition by coupling rate-limiting cell cycle proteins to the translational capacity of the cell. In the budding yeast, S. cerevisiae, cyclin CLN3 (also known as YHC3) contains an upstream open reading frame in the 5' UTR that slows its translation in G1 under poor growth conditions. Similarly, during G2 in the fission yeast, Schizosaccharomyces pombe, accumulation of CDC25 is disproportionately affected by defects in translation. Perhaps the translation of Boule, along with a few other meiotic cell cycle regulators, is disproportionately affected when translation is compromised in spermatocytes. Although this should be investigated, this simpler model does not explain the aberrant accumulation of Rux and the nuclear sequestration of Cyclin A that was observed (Franklin-Dumont, 2007 and references therein).

The defects in differentiation in eIF4G2 mutants are not secondary to the meiotic block, because several cell cycle mutants fail to divide but still undergo substantial post-meiotic differentiation. Several spermatid differentiation genes, such as don juan and fuzzy onions, are transcribed in primary spermatocytes under the control of spermatocyte arrest genes. Translational control delays the accumulation of their protein products. This delay is functionally relevant, because precocious don juan accumulation leads to sterility. In principle, then, the lack of significant differentiation in eIF4G2 mutants could simply be due to a more pronounced translational delay for key differentiation genes. Alternatively, the block in differentiation might reflect a direct effect of the proposed growth checkpoint. Consistent with either model, the accumulation of the mitochondrial fusion protein Fuzzy onions is delayed, although this was not timed precisely. It is expected that other differentiation targets will also be abnormally delayed in eIF4G2 mutants (Franklin-Dumont, 2007).

There are striking parallels to the role of eIF4G2 during spermatogenesis in other organisms. For instance, there are also two major isoforms of eIF4G in Caenorhabditis elegans, encoded by ifg-1. When the longest isoform was depleted from the germ line, oocytes arrested in meiosis I (B. D. Keiper, personal communication to Franklin-Dumont, 2007). The requirement for ifg-1 in spermatogenesis has not yet been examined. However, one of the five isoforms of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA interference against ife-1 results in delayed meiotic progression, and in defective sperm, in both hermaphrodites and males (Amiri, 2001). Furthermore, mouse testes carrying the Y chromosome deletion Spy (also known as Eif2s3y-Mouse Genome Informatics) have a meiotic arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome Informatics) function (Mazeyrat, 2001). Taken together, these examples suggest that translational control, and therefore possibly growth control, is a common theme for meiotic cycle cells (Franklin-Dumont, 2007).


The Drosophila boule gene is expressed exclusively in the male germline. Boule localization was examined in flattened germ cell preparations (testis squashes) by immunocytochemistry. In wild-type testes, Boule is first detected at the beginning of the spermatocyte growth phase, following completion of the premeiotic S phase. Boule staining is diffuse at this stage. However, as the primary spermatocytes mature, Boule becomes highly concentrated in nuclei. Within these nuclei, Boule protein staining is not uniform but is instead consistently restricted to a crescent-shaped region. To define the nature of the subnuclear localization of Boule, the Boule staining pattern was compared to prominent nuclear structures. Comparison with chromatin localization, as assayed with the DNA-binding dye Hoechst, indicates that the Boule crescent is near, but not coincident with, one of the three readily detectable chromosome pairs. The associated chromosomes have been identified as the XY pair, based on the distinct morphology of this chromosome set relative to the autosomes. In most cells, Boule staining does not overlap with that of the XY pair, but is very frequently found nearby, usually at the periphery of the nucleus (Cheng, 1998).

Since the X and Y chromosomes contain the rRNA loci and are therefore closely associated with the nucleolus, phase-contrast microscopy was used to determine the location of Boule relative to this organelle. The spermatocyte nucleolus appears as a roughly spherical, phase-dense structure within the nucleus. The crescent-shaped region of Boule staining very often abuts and embraces the nucleolus, but is not coincident with it. The distinctive Boule crescent thus represents a perinucleolar pattern of protein localization. Boule nuclear localization persists until the end of the spermatocyte growth phase. At the time of nucleolar breakdown, prior to meiosis, Boule is in the cytoplasm rather than the nucleus. As a result, during meiosis Boule is excluded from the chromatin-containing region and is largely cytoplasmic. Boule remains cytoplasmic in postmeiotic spermatids, where it appears to be uniformly distributed. Boule localization in spermatocytes is thus biphasic, being first nuclear and then cytoplasmic. Boule localization was examined in twine mutant spermatocytes, which fail to activate the cell cycle oscillator and hence do not undergo the meiotic divisions. Boule undergoes a wild-type transition from the nucleus to the cytoplasm in the twine mutant. It is concluded that the translocation of Boule to the cytoplasm is not dependent on meiotic entry (Cheng, 1998).

Nuclear localization was dependent on RNA and on the presence of the Y chromosome. Boule nuclear localization is absent from primary spermatocytes in samples treated with RNase. However, the cytoplasmic localization of Boule in postmeiotic onion stage spermatids is unaffected by RNase treatment, indicating that nuclear but not cytoplasmic Boule localization is primarily dependent on RNA (Cheng, 1998).

Deletion of the Y chromosome ks-1 fertility locus eliminates Boule nuclear localization, although it does not perturb entry into meiosis. Based on these observations it is proposed that Boule acts in the cytoplasm to regulate the stability or translation of messenger RNA encoding an essential meiotic factor. The Y chromosome encodes six fertility loci, each spanning four or more megabases of DNA, that are heavily transcribed in the primary spermatocyte. Indeed, several of the resulting lampbrush chromosome structures, or Y loops, are visible by phase microscopy at the nuclear periphery of un-stained primary spermatocytes. It was hypothesized, therefore, that Boule might bind to one or more Y loop structures. To compare Y loop localization with that of Boule, testis squashes were immunostained with an antibody that recognizes Y loop associated antigens in Drosophila chromatin. This antiserum recognizes a subset of the Y loops, predominantly the antigen associated with the kl-5 and to a lesser extent the ks-1 fertility factors. Like Boule, the Y loop antigens occupy a crescent-like region that is often adjacent to the nucleolus. In flattened spermatocytes double stained with anti-Boule and anti-Y loop sera, Boule and the Y loop antigens have been found localized to neighboring regions that occasionally overlap. The similarity and close spatial association of the localization patterns suggests that Boule associates with Y loops (Cheng, 1998).

To delineate the region of the Y required for Boule nuclear localization, a deletion analysis was carried out. Four of the Y chromosome fertility factors lie on the long arm (kl-1, kl-2, kl-3, kl-5) and two on the short arm (ks-1 and ks-2), with numbering on each arm ordered from proximal to distal. Eight distinct sets of Y chromosome synthetic deficiencies were analyzed for alterations in Boule staining. Only the three deficiencies affecting ks-1 disrupt the nuclear localization of Boule. In all three such cases Boule is predominantly cytoplasmic in primary spermatocytes. Staining in later spermatids is not affected by any of the deficiencies. These results indicate that Boule nuclear localization depends on the ks-1 region of the Y. However, since the ks-1-dependent localization of the RNA binding protein RB97D is wild-type in a boule mutant, the Y loop structure derived from the ks-1 region is not substantially altered in the absence of Boule (Cheng, 1998).

Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning

Drosophila mushroom body (MB) γ neurons undergo axon pruning during metamorphosis through a process of localized degeneration of specific axon branches. Developmental axon degeneration is initiated by the steroid hormone ecdysone, acting through a nuclear receptor complex composed of USP (ultraspiracle) and EcRB1 (ecdysone receptor B1) to regulate gene expression in MB γ neurons. To identify ecdysone-dependent gene expression changes in MB γ neurons at the onset of axon pruning, laser capture microdissection was used to isolate wild-type and mutant MB neurons in which EcR (ecdysone receptor) activity is genetically blocked, and expression changes were analyze by microarray. Several molecular pathways that are regulated in MB neurons by ecdysone were identified. The most striking observation is the upregulation of genes involved in the UPS (ubiquitin-proteasome system), which is cell autonomously required for γ neuron pruning. In addition, the function was characterized of Boule, an evolutionarily conserved RNA-binding protein previously implicated in spermatogenesis in flies and vertebrates. boule expression is downregulated by ecdysone in MB neurons at the onset of pruning, and forced expression of Boule in MB γ neurons is sufficient to inhibit axon pruning. This activity is dependent on the RNA-binding domain of Boule and a conserved DAZ (deleted in azoospermia) domain implicated in interactions with other RNA-binding proteins. However, loss of Boule does not result in obvious defects in axon pruning or morphogenesis of MB neurons, suggesting that it acts redundantly with other ecdyonse-regulated genes. A novel function is proposed for Boule in the CNS as a negative regulator of developmental axon pruning (Hoopfer, 2008).

To identify genes that regulate developmental axon pruning, DNA microarrays were used to analyze ecdysone-dependent gene expression changes in MB neurons at the onset of metamorphosis. This analysis identified 1038 genes that show ecdysone-dependent expression in MB neurons at the onset of neuronal remodeling at 0 h APF, or in the early steps of axon pruning at 5 h APF. Approximately 32% of these were previously identified as being regulated by ecdysone at the onset of metamorphosis in the whole animal or the brain. The large number of genes unique to these data set supports the assertion that there has been enrichment for MB specific ecdysone-regulated gene expression changes (Hoopfer, 2008).

Distinct functional classes of genes are differentially regulated by ecdysone during MB axon pruning. These classes give insight into the molecular pathways that regulate neuronal remodeling. For example, the upregulation of genes encoding regulators or structural constituents of the cytoskeleton provides candidate molecules that may be involved in remodeling the axon cytoskeleton during pruning. Also the differential regulation of several genes involved in synaptic transmission is described, including those involved in the synthesis and degradation of neurotransmitters (NTs) and receptors for excitatory and inhibitory NTs. This may reflect developmental changes in the functional properties of the MB neurons, or a response to pruning of their presynaptic partners. Given that little is known about the physiological properties of larval MB neurons, these genes may provide insight into potential functional differences between larval and adult MB neurons (Hoopfer, 2008).

Of particular relevance to axon pruning is the upregulation of genes encoding components of the UPS, because UPS activity is cell-autonomously required in various paradigms of Drosophila neuronal pruning. Interestingly, genes in every step of UPS-mediated protein degradation are upregulated in an ecdysone-dependent manner. What is the functional significance of this transcriptional regulation for axon pruning? One possibility is that UPS function is increased to deal with an increased load of proteins that need to be degraded during pruning. A similar transcriptional coregulation of proteasome genes and the 20S maturase (Pomp in Drosophila) was seen as observed in cultured Drosophila and mammalian cells in response to proteasome stress. Additionally, an upregulation was seen of the Drosophila homolog of the yeast ubiquitin-specific protease Ubp6 (CG5384), which is associated with the proteasome and has been shown to regulate ubiquitin homeostasis by preventing the degradation of ubiquitin. Thus, MB γ neurons exhibit ecdysone-mediated induction of genes that may serve to increase overall UPS-mediated degradation (Hoopfer, 2008).

An ecdysone-dependent upregulation of genes was seen encoding components of the UPS that regulate the target specificity of degradation such as ubiquitin ligases (E3s). Interestingly, multiple genes involved in programmed cell death, which have been shown to be involved in dendrite pruning in Drosophila sensory neurons, such as effete, thread, and Ice, are upregulated by ecdysone in MB neurons at the onset of metamorphosis. However, genetic analysis suggests that none of these genes is required for axon or dendrite pruning in MB γ neurons by itself. Thus, the molecular program used for pruning may differ by cell type, or may be more redundant in the CNS. Identification of the specific ubiquitin ligases and their substrates that are involved in MB pruning should yield insight into how these molecular programs differ. The ubiquitin ligases identified in microarray analysis are attractive candidates for future investigation (Hoopfer, 2008).

boule was identified in two independent screens for genes involved in MB γ neuron pruning. Boule is downregulated in MB neurons by ecdysone at the onset of axon pruning, and overexpression of Boule in γ neurons is sufficient to inhibit axon pruning. Thus, it is proposed that Boule acts as a negative regulator of γ neuron pruning. How does Boule function to inhibit MB γ neuron pruning? Boule contains a highly conserved RNA-binding domain, and has been proposed to regulate meiotic entry during spermatogenesis by stimulating translation of the Drosophila Cdc25-type phosphatase twine through binding to the untranslated regions of twine mRNA. By using multiple insertions of UAS-bolPM1::FLAG to express BolPM1 at similar levels as the wt Bol-A transgenic protein, it was shown that Bol-A blocks axon pruning to a greater extent than BolPM1; however, increased expression of BolPM1 can block axon pruning. A possible explanation for this dosage-dependent difference in the phenotype of BolPM1 may be attributable to the nature of the point mutation. A similar point mutation in tra2 was shown in vitro to decrease RNA-binding specificity, without affecting RNA-binding affinity. Thus, at low levels, BolPM1 may no longer efficiently interact with its RNA targets because of nonspecific interactions, but at higher levels binding may be saturated resulting in an inhibition of pruning (Hoopfer, 2008).

Whereas a mutation in the RNA-binding domain reduces the ability of Boule to inhibit axon pruning, deletion of the DAZ domain abolishes the ability of Boule to block axon pruning. Biochemical analyses of vertebrate DAZ-like (DAZL) proteins have shown that the DAZ domain is essential for translational stimulation by mediating interactions with poly(A) binding proteins. Indeed, mouse DAZL protein has been shown to associate with poly(A)-bound polyribosomes in mouse testes. In addition, human Boule can associate with other RNA-binding proteins, such as Pumilio-2, through its DAZ domain. Thus, the results suggest that Drosophila Boule may inhibit axon pruning by positively regulating the translation of RNAs through an interaction with poly(A)-binding protein or possibly other RNA-binding proteins (Hoopfer, 2008).

Loss-of-function data using a null allele of boule indicate that, whereas Boule expression is sufficient to inhibit γ neuron pruning, loss of Boule expression in MB neurons is not sufficient to initiate axon degeneration in the absence of other factors. This suggests that Boule acts redundantly with other positive regulators of axon pruning that are induced by ecdysone signaling, such as the UPS. Although the possibility that the overexpression of Boule causes a neomorphic phenotype cannot be ruled out, the fact that Boule is expressed in MB neurons and downregulated by ecdysone signaling makes this possibility less likely. Regardless, its gain-of-function phenotype suggests that Boule must perturb the genetic program that regulates MB pruning; thus, the identification of the RNA targets of Boule in MB neurons should identify other molecular players in axon pruning. In the testes, Boule regulates translation of twine mRNA. twine expression was analyzed in pupal MB neurons using a twine-lacZ reporter that faithfully reflects twine translation in the testes, and no twine expression was seen in wt or Boule-overexpressing MB neurons, suggesting the Boule has novel targets in MB neurons (Hoopfer, 2008).

In summary, this study represents the first comprehensive analysis of the transcriptional program induced by ecdysone in a specific population of neurons in the Drosophila brain. The results provide insight into the genetic program that underlies neuronal remodeling. Several molecular pathways were identified that raise interesting hypotheses concerning the mechanisms that regulate both the morphological and functional remodeling of neurons during development. Several genes encoding ubiquitin ligases, regulators of cytoskeletal dynamics, and components of synaptic transmission are promising candidates for future investigation. Importantly, the transcriptional upregulation of UPS components by EcR provides a mechanistic link between ecdysone regulation and UPS activity in axon pruning. Last, the RNA-binding protein Boule was identified in two independent screens for genes involved in MB axon pruning. The function of Boule as a negative regulator of axon pruning suggests that, in addition to the transcriptional regulation of axon pruning by ecdysone signaling, Boule may represent an important point of posttranscriptional regulation for the initiation of axon pruning (Hoopfer, 2008).


Spermatocytes are formed in boule mutants, but fail to undergo meiotic divisions. Comparison of the localization of Cyclin A in boule mutant and in wild-type germ cells supports the conclusion that the meiotic prophase is normal in boule mutants. Although the meiotic prophase appears wild type in boule mutant germ cells. subsequent stages are aberrant. Cyclin A is exclusively cytoplasmic in the extended premeiotic G2, and enter the nucleus at the transition between G2 and M phases only to be degraded rapidly after nuclear translocation, yet Cyclin A persists in boule mutants (Eberhart, 1996).


daz-1: a C. elegans boule homolog

daz-1, the single DAZ homolog in the nematode Caenorhabditis elegans has been identified and characterized. Loss of daz-1 function causes sterility in hermaphrodites, by blocking oogenesis at the pachytene stage of meiosis I. Epistasis analysis suggests that this gene executes its function after gld-1, which governs the early pachytene stage in the oogenic pathway. Spermatogenesis does not appear to be affected in daz-1 hermaphrodites. Males defective in daz-1 produced sperm fully competent in fertilization. Analysis employing sex-determination mutants indicates that the daz-1 function is required for meiosis of female germline regardless of the sex of the soma. Transcription of daz-1 is restricted to the germline, starting prior to the onset of meiosis and is most conspicuous in cells undergoing oogenesis. Thus, daz-1 in C. elegans is an essential factor for female meiosis but, unlike other DAZ family members so far reported, it is dispensable for male meiosis (Karashima, 2000).

DAZ family genes are conserved from nematode to mammals. However, no DAZ homolog has been found in unicellular organisms; this includes meiosis-proficient eukaryotic microbes like budding and fission yeast as well as bacteria. This may imply that the emergence of the DAZ family gene in the course of evolution was associated with the increase of complexity in the mechanisms of meiosis in multicellular organisms. All of the DAZ family genes so far investigated are expressed exclusively in the germline, but their sex-specificity is not identical. The DAZ family genes in Drosophila and C. elegans are required for gametogenesis in one sex: Drosophila boule for spermatogenesis (Eberhart, 1996) and C. elegans daz-1 for oogenesis. Murine Dazla is required for both oogenesis and spermatogenesis, but differently: in Dazla-deficient mice, oogenesis appears to be normal until it is arrested at the pachytene stage, hence mimicking the situation in C. elegans, whereas the male germline generates fewer germ cells, which do not proceed beyond the spermatogonial stage. It is unknown whether the human counterpart of murine Dazla, namely DAZLA/DAZH, is involved in gametogenesis. A deletion of the human DAZ cluster on Y chromosome results in a wide range of spermatogenic deficiency. This variability may reflect the functional redundancy and divergence between DAZ and DAZLA/DAZH (Karashima, 2000).

How have the DAZ family genes acquired such versatile sex-specificity during evolution? Yeast has only one mode of meiosis, i.e., diploid cells generate four spores. In contrast, higher organisms produce asymmetrical gametes. To regulate spermatogenesis and oogenesis independently, each program would require a different mode of meiosis: these two modes should be at least partly distinct from one another. Thus, it is assumed that multicellular organisms have developed two types of mechanisms for meiotic regulation, one of which uses the DAZ family gene at the pachytene stage and the other which does not. Meanwhile, strategies for sex determination are strikingly divergent among species, with no overlapping molecular mechanisms being discovered in C. elegans, Drosophila and mammals. Taken together, during the evolution from a primitive multicellular organism, some organisms may have employed the DAZ-dependent meiosis for spermatogenesis and the DAZ-independent meiosis for oogenesis, whereas others did vice versa. Hopefully more extensive analysis of the DAZ family genes and more intensive analysis of their molecular function will prove or disprove the validity of this hypothesis (Karashima, 2000).

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. Mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, it is proposed that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis (Maruyama, 2005).

Based on the sequence similarity of the RNA-recognition motifs, the DAZ family has been divided into two subgroups, namely, BOULE and DAZL. It was inferred that the BOULE group is the more ancient, that the DAZL group derived from BOULE by duplication in the ancestor of vertebrates, and that DAZ, which belongs to the DAZL group, was generated in primates by duplication of DAZL. The BOULE members other than C. elegans DAZ-1, i.e., Drosophila boule and murine and human BOULE, are expressed in testis but neither ovary nor primordial germ cells. In contrast, all the DAZL members appear to be expressed in both male and female germlines since the primordial germ cells stage. C. elegans DAZ-1 is undetectable in germ cell precursors in the embryo but emerges in both male and female germ cells as they start proliferation at the early larval stage. This expression profile is rather similar to that of the DAZL subgroup. In addition, the function of C. elegans DAZ-1 in vivo is apparently female specific, contrasting with the male specificity of Drosophila Boule. Thus, the expression profiles and the loss-of-function phenotypes of the DAZ family members do not necessarily correlate with the subgrouping that relies on sequence similarity. This divergence of the in vivo function even within the BOULE subgroup implies that the role of the DAZ family members might have been subjected to rapid modification in the course of evolution. However, because some DAZ members can substitute for others in different species, at least partially, they appear to maintain a common biochemical function, presumably as translational regulators utilizing their conserved RNA-binding activity. Further characterization of DAZ family members, including identification of the downstream target genes, is awaited to understand how this family controls germline development and how it has evolved (Maruyama, 2005).

Fish Boule homologs

In many species, DAZ homologous genes encode RNA-binding proteins containing two conserved motifs, namely the RNA-recognition motif (RRM) and the DAZ motif. Genetic analysis and gene disruption studies have demonstrated that DAZ family proteins play important roles in gametogenesis. However, little is known about the biochemical functions of DAZ family proteins. Using in vitro selection and UV-crosslinking experiments, the sequence 'GUUC' was identified as the target RNA sequence of zebrafish DAZ-like protein (zDAZL). In transfection experiments, zDAZL protein activates translation in a manner dependent on the binding sequence in the 3'UTR of the Drosophila twine gene or zDazl gene. Moreover, it is highly likely that the zDAZL protein associates with polysomes through the DAZ motif in vivo, and that the association with polysomes is indispensable for translational activation. This is the first report that the DAZ family protein directly promotes the translation of the target mRNAs in vertebrates. This study provides important insights into the molecular mechanisms underlying the post-transcriptional regulation of DAZ family proteins in gametogenesis (Maegawa, 2002).

Amphibian Boule homologs

A localized RNA component of Xenopus germ plasm has been identified. This RNA, Xdazl (Xenopus DAZ-like), encodes a protein homologous to human DAZ (Deleted in Azoospermia), vertebrate DAZL and Drosophila Boule proteins. Similarity among the DAZ homologs is high: Xdazl and DAZ have 42% identical residues overall, and Xdazl shares approximately 60% identical residues with mouse and human Dazl proteins. Similarity to the Boule protein is less, with 27% of the residues remaining conserved from flies to frogs. Xdazl also contains some residues corresponding to the 24 amino acid DAZ repeat, although the Xdazl protein has a three amino acid insertion, AIQ, in the middle of the motif. In general, the proteins appear well conserved within the RNP domain and more divergent at the amino terminus and in the C-terminal domain. At the nucleotide level, Xdazl is most closely related to the murine Dazl gene. In a stretch of 800 nucleotides constituting most of the coding region, the two genes are approximately 70% identical overall. Also, the sequences of many inferred exon boundaries are highly conserved from frog to man. The high amino acid and nucleotide sequence conservation among the DAZ members suggests that the genes have evolved from a common ancestor and may be involved in similar functions in all organisms (Houston, 1998).

Xdazl RNA is detected in the mitochondrial cloud and vegetal cortex of oocytes. In early embryos, the RNA is localized exclusively in the germ plasm. Consistent with other organisms, Xdazl RNA is also expressed in the spermatogonia and spermatocytes of frog testis. To determine if the RNA is present during specific stages of spermatogenesis, sections of adult and juvenile testis tissue were hybridized with DIG-labeled antisense RNA probes. In both the adult (and juvenile), Xdazl RNA is found in discrete spermatocysts. The majority of Xdazl-expressing cells have been identified as spermatocytes. The observation of metaphase plates in some of the spermatocyte cysts confirms that Xdazl RNA is expressed in the frog testis in the cells undergoing meiotic cell division. Some staining is also detected in spermatogonia; however, Xdazl RNA is consistently absent in spermatids and mature sperm. Proteins in the DAZ-family contain a conserved RNP domain, implying an RNA-binding function. Xdazl can function in vitro as an RNA-binding protein. To determine if the function of Xdazl in spermatogenesis is conserved, the Xdazl cDNA was introduced into boule flies. This resulted in rescue of the boule meiotic entry phenotype, including formation of spindles, phosphorylation of histone H3 and completion of meiotic cell division. Overall, these results suggest that Xdazl may be important for primordial germ cell specification in the early embryo and may play a role analogous to Boule in promoting meiotic cell division (Houston, 1998).

Germ plasm is morphologically similar in all organisms where it is found and is typically composed of a fibrillar 'germinal cytoplasm', electron-dense germinal granules, mitochondria and ribosomes. In Xenopus, the germ plasm is present in eggs as numerous discrete islands at the vegetal pole. These islands aggregate after fertilization, a process that requires a kinesin-like protein, Xklp-1. Germ plasm is segregated unequally during cleavage stages until gastrulation, at which time the germ plasm becomes perinuclear and is divided equally among daughter cells. During subsequent embryogenesis, primorial germ cells (PGCs), carrying the germ plasm, remain in the endoderm and are thought to undergo 2-3 cell divisions. Around stage 32/33 (late tailbud stage), the PGCs begin to migrate dorsally through the lateral endoderm. By early tadpole (stage 40), the PGCs accumulate in the dorsal crest of the posterior endoderm and are subsequently incorporated into the lateral plate mesoderm that forms the dorsal mesentery. In later stages, PGC migration continues to the dorsal body wall and then laterally to the forming genital ridges (Houston, 2000).

In Xenopus, several RNAs have been found localized to the germ plasm but none of these have previously been tested for roles in PGC specification. Several of these RNAs are similar to important Drosophila pole plasm components; Xcat2 encodes a Nanos homolog and Xlsirts encodes an untranslated RNA. Xdazl is expressed in the mitochondrial cloud of stage I oocytes, the source of germ plasm material, and remains expressed in the germ plasm until the neurula stage. Xdazl RNA is also abundantly expressed in germ cells of the testis, but not in any of the somatic tissues. Xdazl encodes an RNA-binding protein and is highly related to genes of the Deleted in Azoospermia (DAZ) (Houston, 2000 and references therein).

Focused upon was the question of whether depletion of maternal Xdazl could cause a deficiency in histologically identifiable PGCs in tadpoles. Xdaz-1 was depleted from oocytes by Xdaz-1 antisense mRNA. Xdazl-depleted oocytes were fertilized and the embryos were raised to the tadpole stage (stage 43/44). PGC numbers in Xdazl-depleted embryos are greatly reduced. In many cases, PGCs are completely eliminated. It was asked whether there were PGCs present in the stage 40 embryos that lacked Xpat (a PGC-specific molecular marker) RNA. Xdazl-depleted stage 40 embryos were shown to lack Xpat RNA and cells with the morphological features of PGCs could not be found. This observation suggests that PGC migration does not occur in the absence of Xpat RNA. Additionally, the lack of Xpat-containing cells remaining in the gut indicates that PGC migration is not merely delayed and that residual PGCs do not persist in the endoderm of Xdazl-depleted embryos (Houston, 2000).

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, DAZ-like sequence was cloned from axolotls and termed Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells (Johnson, 2001).

DAZ, a Y chromosome gene associated with azoospermia

Deletions of portions of the Y chromosome long arm have been detected in 12 of 89 men with azoospermia (no sperm production). No Y deletions were detected in their male relatives or in 90 other fertile males. The 12 deletions overlap, defining a region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. No evidence has been found of YRRM genes, recently proposed as AZF candidates, in the AZF region. The region contains a single-copy gene, DAZ (Deleted in AZoospermia), which is transcribed in the adult testis and appears to encode an RNA binding protein (Reijo, 1995).

Deletion of the Azoospermia Factor (AZF) region of the human Y chromosome results in spermatogenic failure. While the identity of the critical missing gene has yet to be established, a strong candidate is the putative RNA-binding protein DAZ (Deleted in Azoospermia). The mouse homolog of DAZ is described. Unlike human DAZ, which is Y-linked, in mouse the Dazh (DAZ homolog) gene maps to chromosome 17. Nonetheless, the predicted amino acid sequences of the gene products are quite similar, especially in their RNP/RRM (putative RNA-binding) domains, and both genes are transcribed predominantly in the testis; the mouse gene is transcribed at a lower level in ovaries. Dazh transcripts were not detected in testes of mice that lack germ cells. In testes of wildtype mice, Dazh transcription is detectable 1 day after birth (when the only germ cells are prospermatogonia), increases steadily as spermatogonial stem cells appear, plateaus as the first wave of spermatogenic cells enters meiosis (10 days after birth), and is sustained at this level thereafter. This unique pattern of expression suggests that Dazh participates in differentiation, proliferation, or maintenance of germ cell founder populations before, during, and after the pubertal onset of spermatogenesis. Such functions could readily account for the diverse spermatogenic defects observed in human males with AZF deletions (Reijo, 1996).

It is widely believed that most or all Y-chromosomal genes were once shared with the X chromosome. The DAZ gene is a candidate for the human Y-chromosomal Azoospermia Factor (AZF). Multiple copies of DAZ (> 99% identical in DNA sequence) clustered in the AZF region and a functional DAZ homolog log (DAZH) on human chromosome 3 are reported. The entire gene family appears to be expressed in germ cells. Sequence analysis indicates that the Y-chromosomal DAZ cluster arose during primate evolution by (1) transposing the autosomal gene to the Y; (2) amplifying and pruning exons within the transposed gene and (3) amplifying the modified gene. These results challenge prevailing views of sex chromosome evolution, suggesting that acquisition of autosomal fertility genes is an important process in Y chromosome evolution. (Saxena, 1996)

To understand the DAZ gene family and its expression, the DAZ genomic structure and RNA transcripts in numerous males, as well as several DAZ cDNA clones have been analyzed. The results of genomic Southern blot have shown that each male contains multiple DAZ genes with varying numbers of DAZ repeats, and that the copy number of the DAZ repeats are polymorphic in the population. The presence of multiple species of DAZ transcripts with different copy number and the arrangement of the DAZ repeats in an individual suggest that more than one DAZ gene is transcribed. The existence of multiple functional DAZ genes complicates the analysis of genotype/phenotype correlations among males with varying sperm counts (Yen, 1997).

The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, has created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provides a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences have been observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gives a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. It is therefore hypothesized that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55,000 to 200,000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations has showen that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race (Agulnik, 1998).

Defects in human germ cell development are common and yet little is known of genes required for germ cell development in men and women. The pathways that develop germ cells appear to be conserved broadly, at least in outline, in organisms as diverse as flies and humans beginning with allocation of cells to the germ cell lineage, migration of these cells to the fetal gonad, mitotic proliferation and meiosis of the germ cells, and maturation into sperm and eggs. In model organisms, a few thousand genes may be required for germ cell development including meiosis. To date, however, no genes that regulate critical steps of reproduction have been shown to be functionally conserved from flies to humans. This may be due in part to strong selective pressures that are thought to drive reproductive genes to high degrees of divergence. This study investigated the micro- and macro-evolution of the Boule gene, a member of the human DAZ gene family, within primates, within mammals and within metazoans. Sequence divergence of Boule is unexpectedly low and rapid evolution is not detectable. The evolutionary analysis of Boule has been extended to the level of phyla and a human Boule transgene can advance meiosis in infertile boule mutant flies. This is the first demonstration that a human reproductive gene can rescue reproductive defects in a fly. These studies lend strong support to the idea that Boule may encode a key conserved switch that regulates progression of germ cells through meiosis in men (Xu, 2003).

DAZLA, an autosomal gene related to DAZ

The DAZ (Deleted in AZoospermia)and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homolog of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. The human DAZLA gene has been isolated by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologs. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility (Yen, 1996).

A homolog of the human Y linked DAZ gene has been isolated from mouse. This gene, Dazla (Daz like, autosomal), is autosomal, located on chromosome 17 and apparently single copy. The predicted protein is highly homologous to that encoded by the DAZ gene in the N-terminal regions of the two proteins and this homology is not confined to the RNA binding domain. Analysis of its expression pattern by reverse transcription PCR shows that the transcript is only detectable in male and female gonads and that testes lacking germ cells do not contain detectable amounts of transcript. A Y-linked DAZ homolog could not be detected in mouse and these results point to the possibility of a role for autosomal RNA binding proteins in mammalian gametogenesis (Cooke, 1996).

A series of human testis poly(A) cDNA clones has been isolated by cross-hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis reveals an identical nucleotide composition in different 'full-length' clones, suggesting that all were encoded by the same gene. This gene has been mapped to the short arm of chromosome 3 and has been designated SPGYLA (SPGY like autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 has revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present that is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homolog of Dazla and boule (Shan, 1996).

The DAZLA (DAZ Like Autosomal) gene on human chromosome 3 shares a high degree of homology with the DAZ (Deleted in AZoospermia) gene family on the Y chromosome, a gene family frequently deleted in males with azoospermia or severe oligospermia. The involvement of both DAZ and DAZLA in spermatogenesis is suggested by their testis-specific expression and their homology with a Drosophila male infertility gene, boule. Whereas male infertility resulting from deletion of the DAZ genes on the Y chromosome occurs sporadically, that due to a defective DAZLA gene is expected to be inheritable. The fraction of males with idiopathic azoospermia or oligospermia that harbor mutations in the DAZLA gene remains unknown. As a prerequisite for mutation screening, the genomic structure of the DAZLA gene was elucidated and found to consist of 11 exons spanning 19 kh. The exon/intron boundaries are conserved between DAZ and DAZLA. The 5' end of both genes are hypomethylated in spermatozoa but not in leukocytes or placenta, consistent with the expression pattern of the genes. The genomic structure of DAZLA paves the way for mutation detection in families with autosomal recessive male infertility (Chai, 1997).

RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, because they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homolog is essential for spermatogenesis in Drosophila. Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells (Ruggiu, 1997).

The human homolog of the mouse germ cell-specific transcript Tpx2, which maps to mouse chromosome 17, has been isolated. Sequence analysis shows that the human gene is part of the DAZ (Deleted in Azoospermia) family, represents the human homolog of the mouse Dazla and Drosophila boule genes, and is termed DAZLA. Like Dazla and boule, DAZLA is single copy and maps to 3p25. This defines a new region of synteny between mouse chromosome 17 and human chromosome 3. Unlike DAZ, which has multiple DAZ repeats, DAZLA encodes a putative RNA-binding protein with a single RNA-binding motif and a single DAZ repeat. DAZLA is more closely related to Dazla in the mouse than to the Y-linked homolog DAZ (88% identity overall with mouse Dazla compared to 76% identity with the human DAZ protein sequence). Southern blot analysis showed that DAZLA is autosomal in all mammals tested and that DAZ has been recently translocated to the Y chromosome, sometime after the divergence of Old World and New World primates. To investigate the evolutionary relatedness of DAZLA and DAZ further, their partial genomic structures were obtained and compared. This has revealed that the genomic organization of both genes in the 5' region is highly conserved. DAZLA is a new member of the DAZ family of genes, which is associated with spermatogenesis and male sterility. Familial cases of male infertility in humans show an autosomal recessive mode of inheritance. It is possible that some of these families may carry mutations in the DAZLA gene (Seboun, 1997).

The DAZ gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. DAZL1 and DAZ bind similarly to various RNA homopolymers in vitro. An antibody against the human DAZL1 was used to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. These results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation (Tsui, 2000).

DAZ is an RNA-binding protein encoded by a region on the Y chromosome implicated in infertility, and DAZ-like (Dazl) proteins are master regulators of germ line gene expression in all animals. In mice Dazl is expressed only in germ cells and is necessary for meiosis. A dual approach was taken to understand the RNA-binding specificity of the Dazl protein: (1) traditional SELEX and (2) a novel tri-hybrid screen. Both approaches led to the same conclusion, namely that Dazl binds oligo(U) stretches interspersed by G or C residues. In a directed tri-hybrid assay the strongest interaction was with the consensus (GUn)n. This motif is found in the 5' UTR of CDC25C whose homologue is thought to be the target of Boule, the Dazl homologue in flies. CDC25C 5' UTR also interacts specifically with Dazl in vitro. The tri-hybrid screen retrieved UTRs of known genes that may be physiological substrates of Dazl (Venables, 2001).

Members of the Pumilio and DAZL family of RNA binding proteins are required for germ cell development in Drosophila, Xenopus, and Caenorhabditis elegans. This study reports identification and characterization of RNA sequences to which PUM2 and DAZL bind. Human PUM2 specifically recognizes the Drosophila Pumilio RNA target (the NRE or Nanos regulator element sequence); single nucleotide changes in the NRE abolished PUM2 binding. Then, coimmunoprecipitation was used to isolate human transcripts specifically bound by PUM2 and DAZL and subsequently those were identified that contain NRE-like sequence elements. The interacting proteins, PUM2 and DAZL, are capable of binding the same RNA target and mRNA sequences bound by both proteins in the 3'UTR of human SDAD1 mRNA were further characterized. Taken together, the results define sequences to which these germ cell-specific RNA binding proteins may bind to promote germ cell development (Fox, 2005).

Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. The domain of BOL that is required for dimerization and for interaction with PUM2 was mapped. BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development (Urano, 2005).

DAZ family proteins transit from nucleus to cytoplasm at mitosis

It has been reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, this study reports that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established (Reijo, 2000).

Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis

The germ cell-specific RNA-binding protein Dazl (deleted in azoospermia-like, mammalian ortholog of Drosophila Boule) is shown to be phosphorylated by MK2 (MAPKAP kinase 2), a stress-induced protein kinase activated downstream of p38 MAPK. Phosphorylation of human Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein (PABP), resulting in reduced translation of Dazl-regulated target RNAs. Transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule. These results suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl (Williams, 2016). P>


Search PubMed for articles about Drosophila boule

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Yen, P. H., Chai, N. N. and Salido, E. C. (1997). The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions. Mamm. Genome 8(10): 756-9.

Biological Overview

date revised: 20 June 2005

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