InteractiveFly: GeneBrief

virilizer: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

virilizer (vir) influences sex determination in Drosophila. Vir is a putative splicing factor involved in the post-transcriptional regulation of Sex lethal (Sxl). The regulation of Sxl transcription and translation is complex and occurs in two steps. First, activation of the gene is transcriptional and relies on an establishment promoter (P_e), which is transcribed only around blastoderm stage in response to a female X:A ratio (XX:AA). Transcripts derived from P_e give rise to early Sxl protein. Transcription from this promoter ends shortly after blastoderm stage when a constitutive promoter (P_m) becomes active in both sexes. From then on, Sxl expression is post-transcriptionally regulated. In the presence of early Sxl, pre-mRNA derived from P_m is female-specifically spliced by skipping exon 3. This exon contains STOP codons in all three reading frames, and therefore functional late Sxl cannot be produced if the exon is present in the mRNA. Late Sxl is used continuously to splice Sxl pre-mRNA in the female mode, and an autoregulatory feedback loop is established. Sxl directs exon skipping by cooperatively binding to multiple uridine-rich stretches within and around the regulated exon. A male primary signal (X:AA) does not activate P_e, and early Sxl is not produced. As a consequence, exon 3 is retained in the Sxl mRNAs when P_m becomes active in males (Niessen, 2001 and references therein).

In addition to Sxl itself and vir, two other genes are needed for Sxl autoregulation: sans fille (snf) and female-lethal-2-d [fl(2)d]. Female-specific mutations exist in vir, snf and fl(2)d that affect the expression of Sxl, but most alleles are lethal to both sexes, suggesting also a more general vital role. snf encodes a nuclear protein with functional and sequence similarity to the mammalian U1A and U2B" snRNP proteins. Sxl physically interacts with SNF via its RRM 1 (RNA recognition motif 1). Therefore, snf is probably directly involved in splice site recognition. fl(2)d encodes a novel nuclear protein with an amino terminal HisGlu-rich domain that is often found in transcriptional regulators. FL(2)D is expressed at all stages of development in both sexes. For virilizer (the subject of this overview) three phenotypic classes of alleles are known. One temperature-sensitive allele, vir^1ts, transforms XX animals into intersexes at the restrictive temperature of 29°; this mutation shows that vir is involved in sex determination. A different allele, vir^2f, is an XX-specific lethal that interferes with dosage compensation and sex determination. Alleles in the third and largest class kill both sexes in the third larval instar, implying a vital function of vir unrelated to sex. In cell clones, the lethal alleles cause sexual transformation of XX cells into male structures, and in trans with vir^2f, they cause female-specific lethality, suggesting that they lack the function required for female-specific expression of Sxl. Indeed, in XX animals mutant for vir^2f, Sxl pre-mRNA is spliced in the male mode, indicating that vir is required for the female-specific splicing of Sxl transcripts (Hilfiker, 1995 and Niessen, 2001 and references therein).

In contrast to the complex effects of vir mutations, the gene itself produces only a single transcript that encodes a large protein. The structure of the putative Vir protein has prompted speculation about the molecular mechanism by which vir affects the expression of Sxl and other vital genes. The prediction of a transmembrane domain, the nuclear localization of Vir, and the domain structure suggest that Vir may be a member of a new class of splice regulators (Niessen, 2001).

Sxl is not the only gene that requires the function of vir. Female-specific expression of transformer and msl-2 depend on vir even when Sxl protein is present. This is seen in XX animals mutant for vir^2f, which can be partially rescued by the constitutive allele Sxl^M4. However, such females are strongly masculinized because the transcripts of tra are not efficiently spliced in the female mode, and their low survival rate points to only partial repression of msl-2 (Hilfiker, 1995). That vir is directly involved in splicing of Sxl and tra pre-mRNA cannot be ruled out, but Vir is not an essential component of the general splicing machinery because even amorphic vir alleles allow growth and differentiation of imaginal discs. Instead, Vir might be used to splice a subset of pre-mRNAs. Among these are the transcripts of Sxl and tra, which are both needed only in females. Another target is the primary transcript of the Ubx gene (Burnette, 1999). In the absence of Vir or FL(2)D, microexons mI and mII are not efficiently included in Ubx transcripts. There are probably other essential genes whose splicing depends on vir and fl(2)d. The sex-unspecific lethality associated with amorphic alleles of vir could be caused by the failure to splice the pre-mRNAs of such targets in the correct manner (Niessen, 2001).

How does vir perform the three functions associated with it, namely, a role in sex determination, in dosage compensation, and in a vital process unrelated to sex? The effects of vir mutation on sex determination and dosage compensation are easily explained by the requirement of vir for the female-specific splicing of transcripts of Sxl, which controls both these processes (Hilfiker, 1995). XX animals mutant for the female-specific allele vir^2f express the gene Sxl in the male mode. Such animals could lack one protein variant that is necessary for female-specific splicing of Sxl pre-mRNA. vir alleles lethal for XX and XY animals might in addition miss another protein variant required for the vital function. Both variants would be missing in the latter class of mutants, which are deficient for the function required for Sxl autoregulation as well as for the vital function. Molecular analysis, however, shows that vir, despite its complex function, produces only a single transcript that probably encodes a single protein. The two XX-specific vir alleles, vir^2f (1283 M-K) and vir^1ts (1423 E-K), cause amino acid substitutions that lie relatively close to one another. These mutations may define a domain in Vir that is essential for female-specific splicing of Sxl pre-mRNAs, but not for the vital function of the protein. This hypothesis implies that a second domain exists that harbors the vital function. However, the genetic data are more in favor of a single protein with a single molecular function. Except for the two female-specific alleles, all the 34 vir mutations isolated so far affect the regulation of Sxl and the vital function. The three lethal alleles (vir⁴, vir²², and vir²³) all cause large carboxy-terminal truncations of Vir. These truncations also remove the region mutated in vir^1ts and in vir^2f: this is consistent with the lethal alleles also being deficient in Sxl autoregulation (Hilfiker, 1995 and Niessen, 2001).

Different threshold requirements might exist for vir in Sxl autoregulation and in the vital process, the former being more sensitive than the latter. The female-specific and the lethal mutations may represent weaker (hypomorphic) and stronger (amorphic) alleles of the gene (Niessen, 2001).

Loss-of-function mutations in vir that cause male-specific splicing of the Sxl pre-mRNA in XX animals suggest a nuclear function for vir in the regulation of Sxl (Hilfiker, 1995). Consistent with this idea, Vir contains an NLS, and a Vir fusion protein locates to the nucleus in Drosophila tissue culture cells. This result, however, does not yet reveal the specific molecular role of the protein. The amino acid sequence of Vir contains a putative transmembrane domain at the amino terminus. Three regions in the protein have a high probability of forming a coiled coil, which, in other proteins, was demonstrated to serve as an interface for homo- or hetero-dimeric binding. Furthermore, a region is found in Vir with similarity to domain 6303 of the ProDom database. This domain is found in several RNA and DNA helicases, in translation initiation factor 2 (IF2) from Borrelia, and in ribonucleoproteins. The member of the family with the highest similarity to Vir within this domain is a human RNA-binding protein (TrEMBL: Q15415) that is thought to be involved in RNA processing or translation. However, there are also striking similarities between Vir and ROG_Human, which is the human hnRNP G. Domain 6303 is present in 4 other hnRNPs as well as hnRNP G: hnRNP A1, D1, R, and U all contain this domain. The proposed functions of these hnRNPs are diverse. hnRNP A1 is involved in RNA splicing, mRNA transport, and telomere biogenesis; hnRNP D1 is thought to be involved in transcription, while hnRNP U has a function in nuclear retention. No functions have yet been assigned to hnRNP G and R (Niessen, 2001 and references therein).

To test if this domain is preferentially present in proteins that interact with RNA, a pattern was derived from domain 6303 and it was used to scan various data banks (using the PatternFind Server at ISREC). Thirty-three proteins were recovered that contained the pattern. Fifteen of them are indeed predicted to interact with RNA (e.g., rRNA methyltransferases, RNA helicases, snRNPs, poly(A)binding proteins) and three are predicted to bind to DNA. Because domain 6303 is also found in proteins without known nucleic acid binding ability and is not present in some proteins that do bind to nucleic acids, it is unlikely that this domain confers binding itself. Rather, it might act as an interface for protein interactions (Niessen, 2001).

The prediction of a transmembrane domain, the nuclear localization of Vir, and the presence of domain 6303 suggest that Vir may be a member of a new class of splice regulators, such as IRE1P, a transmembrane protein that acts as an unconventional splice factor in the unfolded protein response pathway in yeast. However, no sequence homology exists between Vir and IRE1P. The characteristics of Vir led to the speculation that the protein is located in the nuclear membrane where it may mediate mRNA transport. If this were correct, this process could be severely disrupted in strong vir mutants, with mRNAs of vital genes being affected, which would cause sex-unspecific lethality. However, even presumably amorphic alleles are not cell lethal in genetic mosaics (Hilfiker, 1995). In contrast, null alleles of snf, which encodes a component of the general splicing machinery, are incompatible with cell survival. Unlike the strong alleles of vir, the female-specific alleles (vir^1ts and vir^2f) may reduce effectiveness of the nuclear transportation system such that dose-sensitive processes begin to fail. Autoregulation of Sxl is indeed a sensitive system. Even mutations in aspartyl- as well as in tryptophanyl tRNA synthetase of Drosophila can act as maternal modifiers and disrupt the autoregulation of Sxl. However, the HIS-tagged Vir fusion protein is found in the nucleoplasm of Schneider cells and not in the membrane. This finding does not exclude the possibility of membrane insertion because expression might not be physiological in this experiment and staining from excess protein in the nucleoplasm could mask staining at the periphery. In addition, Vir could be localized to the nucleoplasm and also to the membrane in wild type. These two possibilities are compatible with the speculation about a function in nuclear transport (Niessen, 2001).

In summary, vir emerges as a gene that functions in several developmental pathways: sex determination, dosage compensation and vital processes. It seems to achieve this by regulating Sxl and tra, and presumably msl-2, as well as yet unknown target genes required for vital functions in both sexes. The common molecular mechanism by which vir performs these various functions may be its involvement in the process of splicing whereby the splicing of Sxl, and to a lesser extent of tra and msl-2, appears particularly sensitive to malfunction of vir (Hilfiker, 1995).

Chinmo prevents transformer alternative splicing to maintain male sex identity

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform tra^F. In turn, Tra^F alternatively splices dsx to the female isoform dsx^F. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs; cyst stem cells) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. This feminization occurs by transcriptional and post-transcriptional regulation of tra^F. chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). tra^F splicing in chinmo-deficient CySCs leads to the production of Dsx^F at the expense of the male isoform DsxM, and both Tra^F and Dsx^F are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, this study shows that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. This work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of tra^F production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility (Brmai, 2018).

This study shows that that one single factor, Chinmo, preserves the male identity of adult CySCs in the Drosophila testis by regulating the levels of canonical sex determinants. CySCs lacking chinmo lose DsxM expression not by transcriptional loss but rather by alternative splicing of dsx pre-mRNA into dsx^F. These chinmo-mutant CySCs ectopically express Tra^F and Dsx^F, and both factors are required for their feminization. Furthermore, the results demonstrate that tra alternative splicing in cyst cells lacking chinmo is achieved independently of Sxl. Instead, this work strongly suggests that tra^F production in the absence of chinmo is mediated by splicing factors Vir and Fl(2)d. It is proposed that male sex identity in CySCs is maintained by a two-step mechanism whereby tra^F is negatively regulated at both transcriptional and post-transcriptional levels by Chinmo (see Model for adult somatic sex maintenance in the Drosophila somatic gonad). In this model, loss of chinmo from male somatic stem cells first leads to transcriptional upregulation of tra pre-mRNA as well as of vir and fl(2)d. Then the tra pre-mRNA in these cells is spliced into tra^F by the ectopic Vir and Fl(2)d proteins. The ectopic Tra^F in chinmo-deficient CySCs then splices the dsx pre-mRNA into dsx^F, resulting in loss of Dsx^M and gain of Dsx^F, and finally induction of target genes usually restricted to follicle cells in the ovary (Brmai, 2018).

Chinmo has motifs associated with transcriptional repression and its loss clonally is associated with ectopic transcription. One interpretation of the data is that Chinmo directly represses tra, vir, and fl(2)d in male somatic gonadal cells. As the binding site and potential co-factors of Chinmo are not known, future work will be needed to determine whether Chinmo directly regulates expression of these genes. It is also noted that ~50% of chinmo-mutant testes still feminize in the genetic absence of tra or dsx^F. These latter data indicate that Chinmo regulates male sex identity through another, presumably parallel, mechanism that does not involve canonical sex determinants. However, this tra/dsx-independent mode of sex maintenance downstream of Chinmo is not characterized and will require the identification of direct Chinmo target genes (Brmai, 2018).

Previous work has shown that JAK/STAT signaling promotes chinmo in several cell types, including CySCs (Flaherty, 2010). Since JAK/STAT signaling is itself sex-biased and restricted to the embryonic male gonad, it is presumed that activated Stat92E establishes chinmo in male somatic gonadal precursors, perhaps as early as they are specified in the embryo. Because loss of Stat92E from CySCs does not result in an apparent sex transformation phenotype, the interpretation is favored that Stat92E induces expression of chinmo in CySCs but that other sexually biased factors maintain it. One potential candidate is Dsx^M, which is expressed specifically in early somatic gonads and at the same time when Stat92E activation is occurring in these cells. In fact, multiple Dsx^M ChIP-seq peaks were identified in the chinmo locus, suggesting potential regulation of chinmo by Dsx^M. This suggests a potential autoregulatory feedback loop whereby Dsx^M preserves its own expression in adult CySCs by maintaining Chinmo expression, which in turn prevents tra^F and dsx^F production (Brmai, 2018).

Recent studies on tissue-specific sex maintenance demonstrate that while the Sxl/Tra/Dsx hierarchy is an obligate and linear circuit during embryonic development, at later stages it is more modular than previously appreciated. For example, Sxl can regulate female-biased genes in a tra-independent manner. Additionally, Sxl and Tra^F regulate body size and gut plasticity independently of the only known Tra^F targets, ^dsx and fru. Negative regulation of the Tra^F-Dsx^F arm of this cascade is required to preserve male sexual identity in CySCs but unexpectedly is independent of Sxl. Because depletion of Vir or Fl(2)d significantly blocks sex transformation and both are required for tra alternative splicing in the ovary, this work reveals they can alternatively splice tra pre-mRNA even in the absence of Sxl. This is the first demonstration of Sxl-independent, Tra-dependent feminization. These results raise the broader question of whether other male somatic cells have to safeguard against this novel mechanism. Because recent work has determined that sex maintenance is important in systemic functions regulated by adipose tissue and intestinal stem cells, it will be important to determine whether Chinmo represses tra^F in these settings. Finally, since the transcriptional output of the sex determination pathway is conserved from Drosophila (Dsx) to mammals (DMRT1), it is possible that transcriptional regulation of sex determinants plays a similar role in adult tissue homeostasis and fertility in higher organisms (Brmai, 2018).

Role of Hakai in m⁶A modification pathway in Drosophila

N6-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. This study shows that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m⁶A writer components, and Hakai mutants exhibit typical m⁶A pathway defects in Drosophila, such as lowered m⁶A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m⁶A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, it was demonstrated that m⁶A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m⁶A writer. Together, this work not only advances the understanding of mechanism and regulation of the m⁶A writer complex, but also provides insights into how Sxl cooperate with the m⁶A pathway to control its own splicing (Wang, 2021).

There are a variety of chemical modifications on biological macromolecules, such as proteins, nucleic acids, and glycolipids. Like DNA methylation and histone modification, RNA modification represents an extra layer of epigenetic regulatory mechanism. More than 150 chemical modifications in RNA have been discovered, and their biological functions are only starting to be revealed. Chemical modifications of RNA exist in all organisms and for all forms of RNA, including tRNA, rRNA, mRNA, and long noncoding RNA. Common RNA modifications include N6-methyladenosine (m⁶A), N6,2’-O-dimethyladenosine (m⁶A_m), N1-methyladenosine (m¹A), 5-methylcytidine (m⁵C), N4-acetylcytidine (ac⁴C), 7-methylguanosine (m⁷G), and pseudouridine (Ψ). Among them, m⁶A is the most abundant internal modification of mRNA in eukaryotes. Although m⁶A in mRNA was found more than 40 years ago, it was only recently that the field has made extensive progress owing to technological and experimental breakthroughs. By combining m⁶A-specific antibody and high-throughput sequencing, MeRIP-Seq or m⁶A-Seq allows the m⁶A mapping at the whole transcriptome level, thereby providing the possibility to correlate RNA modifications with their biological functions . These and subsequent studies revealed that m⁶A sites contain a consensus motif RRACH (R = G/A; H = U/A/C), and m⁶A peaks are enriched in the 3' untranslated region (UTR) and near the stop codon in yeast and mammals. In Arabidopsis, m⁶A is enriched not only in 3'UTRs and near the stop codon but also in 5'UTRs and around the start codon. In mammalian cells, m⁶A also accumulates in the 5'UTR region in response to stress conditions such as heat shock. The distribution of m⁶A is important since it implies the mechanism by which m⁶A modification regulates its mRNA (Wang, 2021).

Another major breakthrough is the gradual elucidation of the m⁶A modification pathway by biochemical and genetic studies. The m⁶A is deposited by a multicomponent methyltransferase complex ('writers'), mainly recognized by YTH domain-containing 'readers', and can be removed by FTO and ALKBH5 'erasers', although FTO was also indicated as an m⁶A_m demethylase. The key catalytic component of the m⁶A writer complex, Mettl3, was purified and cloned in the 1990s. Since then, studies from yeast, Arabidopsis, Drosophila, and mammalian cells have identified several core components of the writer complex, including Mettl14, WTAP (Fl(2)d), VIRMA (Virilizer), RBM15/15B (Spenito), ZC3H13 (Flacc or Xio), and Hakai. Interestingly, Fl(2)d, Virilizer (Vir), Spenito (Nito), and Xio were first identified from Drosophila sex determination screens and later realized as part of the writer complex. They regulate Drosophila sex determination by controlling the alternative splicing of the master regulatory gene Sex-lethal (Sxl). Recently, Mettl3, Mettl14, as well as the reader Ythdc1, were also shown to be involved in this process. However, the detailed mechanism of how the m⁶A modification cooperates with Sxl protein to modulate its own splicing is still unclear. Thus, Drosophila can serve as a unique system to screen components in the m⁶A pathway and pinpoints a critical role for m⁶A in regulating splicing. Other than Sxl splicing, Drosophila m⁶A genes are highly expressed in the nervous system and exhibit similar wing and behavior defects when mutated. Mutants of several fly m⁶A factors are viable and thus provide an ideal model to study other processes, such as metabolism and immunity, in the future (Wang, 2021).

Hakai, also known as CBLL1, was found as an interacting protein with several m⁶A writer components in proteomic studies. It encodes a RING finger-type E3 ubiquitin ligase and was originally identified as an E-cadherin-binding protein in human cell lines. It was proposed that Hakai ubiquitinates E-cadherin at the plasma membrane and induces its endocytosis, thus playing a negative role post-translationally. Due to the key role of E-cadherin in tumor metastasis, especially epithelial-mesenchymal transition, Hakai has been extensively studied mainly using cell culture and overexpression system, but a previous study using the Drosophila model did not observe an increase of E-cadherin level in Hakai mutants. In Arabidopsis, Hakai mutants show partially reduced m⁶A levels and the mutant phenotypes are weaker than other writer components. Importantly, the in vivo role of Hakai as a core m⁶A writer component has not been studied in any animal species. This study analyzed the role of Hakai in the Drosophila m⁶A modification pathway. The results demonstrated that Hakai is a bona fide member of the m⁶A writer complex, with its mutants showing reduced global m⁶A levels, typical m⁶A mutant phenotypes, and commonly-regulated gene sets. A high-quality fly m⁶A methylome was obtained using stringent MeRIP-seq, discovered a female-specific m⁶A methylation pattern for Sxl mRNA, characterized the role of Hakai in the m⁶A writer complex, and finally revisited the function of Hakai in E-cadherin regulation (Wang, 2021).

m⁶A modification has been known for more than 40 years but has recently gained great attention due to the emergence of technologies to map m⁶A methylome, as well as the identification of the writers, readers, and erasers in this pathway. Since the initial purification of the key methyltransferase Mettl3, other components of the writer complex were gradually identified through biochemical experiments and genetic screens. It is now known that m⁶A writer complex is comprised of multiple components including Mettl3, Mettl14, WTAP, VIRMA, RBM15/15B, ZC3H13. Hakai was first indicated as a WTAP interaction protein and was shown later to be required for full m⁶A methylation in Arabidopsis; however, its role in the m⁶A pathway in animals has not been studied. This study shows that Hakai interacts with other m⁶A writer subunits, and Hakai mutants exhibit characteristic m⁶A pathway phenotypes, such as lowered m⁶A levels in mRNA, aberrant alternative splicing of Sxl and other genes, held-out wings, and flightless flies, as well as reduced m⁶A peaks shared with Mettl3 and Mettl14 mutants in MeRIP-seq. Altogether, these data unambiguously argue that Hakai is the seventh, and likely last core component of the conserved m⁶A writer complex (Wang, 2021).

Each component in the m⁶A writer complex plays a role in mRNA methylation but their exact roles are not well understood. This systematic analysis of several m⁶A writer subunits has provided insights into the mechanism of this important complex. l(2)d, Vir, Hakai, and Flacc were found to form a stable complex, and knocking down either of Fl(2)d, Vir, or Hakai led to the degradation of the other three components. Mettl3, Mettl14, and Nito were not affected by the disruption of Fl(2)d, Vir or Hakai, suggesting that they have separate functions. Knocking down Flacc resulted in less nuclear staining of Fl(2)d, consistent with a role in nuclear localization of the writer complex. Based on these results, a model is proposed for the m⁶A methyltransferase complex. Mettl3 and Mettl14 form a stable heterodimer to catalyze the addition of the methyl group to mRNA. Nito/RBM15 contains three RRM domains and binds to positions adjacent to m⁶A sites, thus may provide target specificity for the m⁶A writer complex. Fl(2)-Vir-Hakai-Flacc form a platform to connect different components and may integrate environmental and cellular signals to regulate m⁶A methylation (Wang, 2021).

Hakai is a potential E3 ubiquitin ligase with an intact C3HC4 RING domain and a C2H2 domain. Its absence led to the degradation, rather than the accumulation of other m⁶A writer subunits, indicating that it may not act as an E3 ubiquitin ligase in this complex. Hakai was initially identified as an E-cadherin binding protein to downgrade its levels CR50, and the role of Hakai in cell proliferation and tumor progression was extensively studied in cell culture. However, the current in vivo analysis using various genetic tools did not find a role of Hakai in E-cadherin regulation. In addition, Hakai appeared as a ubiquitous nuclear protein showing little co-localization with E-cadherin in the membrane. Consistently, Hakai was shown to interact with PTB-associated splicing factor (PSF), a nuclear protein, and to affect its RNA-binding ability. Thus, the role of Hakai in E-cadherin regulation needs to be further investigated using the knockout mouse model and whether Hakai has other substrates for its E3 ligase activity also needs to be determined (Wang, 2021).

Recent emerging studies suggest that m⁶A is involved in numerous developmental processes and human diseases, mainly by regulating mRNA stability, translation, or splicing. Pioneer work has established the framework for the m⁶A pathway in Drosophila. However, only published Drosophila m⁶A methylome was performed in S2R + cells or embryos and was not done against writer mutants. Other than Sxl, few m⁶A target loci have been firmly mapped. By performing MeRIP-seq in wild-type adult flies as well as Mettl3, Mettl14, and Hakai mutants, this study demonstrated that although most m⁶A peaks are distributed in 3'UTRs, the functional peaks responding to the loss of m⁶A writers are mainly located in 5'UTRs. This finding indicates a major difference between Drosophila and mammalian m⁶A methylome, that mainly occurs in 3'UTRs, and is in agreement with a recently published manuscript using miCLIP. Interestingly, LC-MS data show that the overall level of m⁶A modification in Drosophila only accounted for 10-20% of that in mammalian cells. Mettl3 or Mettl14 mutants are embryonic lethal in mice while they develop into adults in flies. It is possible that the m⁶A pathway acquires additional functions during evolution (Wang, 2021).

m⁶A modification in 3'UTRs usually causes mRNA instability and m⁶A in 5'UTRs is linked to translation enhancement. In agreement with the view that functional m⁶A peaks are located in 5'UTRs in Drosophila, this study did not observe an increase in mRNA half-life of m⁶A targets in Mettl3 mutants compared to wild-type. These results imply that the major role of m⁶A modification in Drosophila is not on mRNA degradation, but possibly on translation upregulation, which can be tested by combining ribosome profiling and functional analysis of a single transcript in the future. The current data by combining MeRIP-seq and splicing analysis shed light on how the m⁶A modification contributes to splicing regulation. In all five cases analyzed, four (Dsp1, CG8929, fl(2)d, Aldh-III) in 5'UTRs and one (Sxl) in exon/intron, reduction of m⁶A modification was correlated with enhanced splicing, arguing that the normal role of these modifications might be to repress splicing events nearby (Wang, 2021).

Last but probably the most interesting finding from this work is to demonstrate the female-specific m⁶A modification around Sxl exon3. Sxl is a textbook paradigm to study alternative splicing and has been intensively investigated for more than thirty years. Sxl protein binds to its own mRNA to control the alternative splicing, but its binding sites are located ~200 nucleotides downstream or upstream of the male exon, meaning other regulators should be involved. Recently, the m⁶A modification pathway was shown to modulate Sxl alternative splicing, but the detailed mechanism has not been resolved. The MeRIP-seq data revealed that several m⁶A peaks were deposited only in females on and around Sxl exon3, and they were in the vicinity of Sxl-binding sites. This finding was further validated by independent m⁶A-IP-qPCR and showed that these modifications were reduced in Mettl3 mutant females. This unexpected finding suggests a model that one main function of Sxl may be to recruit the m⁶A writer complex that methylates nearby m⁶A sites. The m⁶A reader Ythdc1 in turn binds to these sites and might interact with the splicing machinery to repress splicing. Future experiments, such as interactions between Sxl and Mettl3/Mettl14, interactions between Ythdc1 and general splicing factors, mapping of the exact m⁶A methylation site in Sxl at the single nucleotide level, comparison of transcriptome-wide binding sites of Sxl with m⁶A modification sites, will be required to firmly prove the model (Wang, 2021).

REGULATION

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

N⁶-methyladenosine (m⁶A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m⁶A methylosome is a complex of nuclear proteins that includes a stable heterodimer [METTL3 (methyltransferase-like 3) and METTL14], WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 (Inducer of meiosis 4) , Mettl14 (Methyltransferase-like 14 ), the Wilms tumour 1-associated protein Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m⁶A in introns in the regulation of alternative splicing remain uncertain. This study shows that m⁶A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout model, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m⁶A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m⁶A reader protein YT521-B decodes m⁶A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m⁶A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m⁶A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression (Haussmann, 2016).

In mature mRNA the m⁶A modification is most prevalently found around the stop codon as well as in 5' untranslated regions (UTRs) and in long exons in mammals, plants and yeast. Since methylosome components predominantly localize to the nucleus, it has been speculated that m⁶A localized in pre-mRNA introns could have a role in alternative splicing regulation in addition to such a role when present in long exons. This prompted an investigation of whether m⁶A is required for Sxl alternative splicing, which determines female sex and prevents dosage compensation in females. A null allele of the Drosophila METTL3 methyltransferase homologue Ime4 was induced by imprecise excision of a P element inserted in the promoter region. The excision allele Δ22-3 deletes most of the protein-coding region, including the catalytic domain, and is thus referred to as Ime4^null. These flies are viable and fertile, but both flightless and this phenotype can be rescued by a genomic construct restoring Ime4. Ime4 shows increased expression in the brain and, as in mammals and plants, localizes to the nucleus (Haussmann, 2016).

Following RNase T1 digestion and ³²P end-labelling of RNA fragments, m⁶A was detected after guanosine (G) in poly(A) mRNA of adult flies at relatively low levels compared to other eukaryotes, but at higher levels in unfertilized eggs. After enrichment with an anti-m⁶A antibody, m⁶A is readily detected in poly(A) mRNA, but absent from Ime4^null flies (Haussmann, 2016).

As found in other systems, and consistent with a potential role in translational regulation, m⁶A was detected in polysomal mRNA, but not in the poly(A)-depleted rRNA fraction. This also confirmed that any m⁶A modification in rRNA is not after G in Drosophila (Haussmann, 2016).

Consistent with the hypothesis that m⁶A plays a role in sex determination and dosage compensation, the number of Ime4^null females was reduced to 60% compared to the number of males, whereas in the control strain female viability was 89%. The key regulator of sex determination in Drosophila is the RNA-binding protein Sxl, which is specifically expressed in females. Sxl positively auto-regulates expression of itself and its target transformer (tra) through alternative splicing to direct female differentiation. In addition, Sxl suppresses translation of msl-2 to prevent upregulation of transcription on the X chromosome for dosage compensation; full suppression also requires maternal factors. Accordingly, female viability was reduced to 13% by removal of maternal m⁶A together with zygotic heterozygosity for Sxl and Ime4 (Ime4^Δ22-3 females crossed with Sxl^7B0 males, a Sxl null allele). Female viability of this genotype is completely rescued by a genomic construct or by preventing ectopic activation of dosage compensation by removal of msl-2. Hence, females are non-viable owing to insufficient suppression of msl-2 expression, resulting in upregulation of gene expression on the X chromosome from reduced Sxl levels. In the absence of msl-2, disruption of Sxl alternative splicing resulted in females with sexual transformations displaying male-specific features such as sex combs, which were mosaic to various degrees, indicating that Sxl threshold levels are affected early during establishment of sexual identities of cells and/or their lineages. In the presence of maternal Ime4, Sxl and Ime4 do not genetically interact (Sxl^7B0/FM7 females crossed with Ime4^null males, 103% female viability. In addition, Sxl is required for germline differentiation in females and its absence results in tumorous ovaries. Consistent with this, tumorous ovaries in Sxl^7B0/+;Ime4^null/+ daughters from Ime4^null females or heterozygous Sxl^7B0 females (Haussmann, 2016).

Furthermore, levels of the Sxl female-specific splice form were reduced to approximately 50%, consistent with a role for m⁶A in Sxl alternative splicing . As a result, female-specific splice forms of tra and msl-2 were also significantly reduced in adult females (Haussmann, 2016).

To obtain more comprehensive insights into Sxl alternative splicing defects in Ime4^null females, splice junction reads were examined from RNA-seq. Besides the significant increase in inclusion of the male-specific Sxl exon in Ime4^null females, cryptic splice sites and increased numbers of intronic reads were detected in the regulated intron. Consistent with reverse transcription polymerase chain reaction (RT–PCR) analysis of tra, the reduction of female splicing in the RNA sequencing is modest, and as a consequence, alternative splicing differences of Tra targets dsx and fru were not detected in whole flies, suggesting that cell-type-specific fine-tuning is required to generate splicing robustness rather than being an obligatory regulator. In agreement with dosage-compensation defects as a main consequence of Sxl dysregulation in Ime4^null mutants, X-linked, but not autosomal, genes are significantly upregulated in Ime4^null females compared to controls (Haussmann, 2016).

Furthermore, Sxl mRNA is enriched in pull-downs with an m⁶A antibody compared to m⁶A-deficient yeast mRNA added for quantification. This enrichment is comparable to what was observed for m⁶A-pull-down from yeast mRNA (Haussmann, 2016).

To map m⁶A sites in the intron of Sxl, an in vitro m⁶A methylation assay was employed using Drosophila nuclear extracts and labelled substrate RNA. m⁶A methylation activity was detected in the vicinity of alternatively spliced exons. Further fine-mapping localized m⁶A in RNAs C and E to the proximity of Sxl-binding sites. Likewise, the female-lethal single amino acid substitution alleles fl(2)d¹ and vir^2F interfere with Sxl recruitment, resulting in impaired Sxl auto-regulation and inclusion of the male-specific exon. Female lethality of these alleles can be rescued by Ime4^null heterozygosity, further demonstrating the involvement of the m⁶A methylosome in Sxl alternative splicing (Haussmann, 2016).

Next, alternative splicing changes was globally analyzed in Ime4^null females compared to the wild-type control strain. A statistically significant reduction in female-specific alternative splicing of Sxl was observed. In addition, 243 alternative splicing events in 163 genes were significantly different in Ime4^null females, equivalent to around 2% of alternatively spliced genes in Drosophila. Six genes for which the alternative splicing products could be distinguished on agarose gels were confirmed by RT-PCR. Notably, lack of Ime4 did not affect global alternative splicing and no specific type of alternative splicing event was preferentially affected. However, alternative first exon (18% versus 33%) and mutually exclusive exon (2% versus 15%) events were reduced in Ime4^null compared to a global breakdown of alternative splicing in wild-type Drosophila, mostly to the extent of retained introns (16% versus 6%), alternative donor (16% versus 9%) and unclassified events (14% versus 6%). Notably, the majority of affected alternative splicing events in Ime4^null were located to the 5' UTR, and these genes had a significantly higher number of AUG start codons in their 5' UTR compared to the 5' UTRs of all genes. Such a feature has been shown to be relevant to translational control under stress conditions. (Haussmann, 2016 and references therein).

The majority of the 163 differentially alternatively spliced genes in Ime4 females are broadly expressed (59%), while most of the remainder are expressed in the nervous system (33%), consistent with higher expression of Ime4 in this tissue. Accordingly, Gene Ontology analysis revealed a highly significant enrichment for genes involved synaptic transmission (Haussmann, 2016).

Since the absence of m⁶A affects alternative splicing, m⁶A marks are probably deposited co-transcriptionally before splicing. Co-staining of polytene chromosomes with antibodies against haemagglutinin (HA)-tagged Ime4 and RNA Pol II revealed broad co-localization of Ime4 with sites of transcription, but not with condensed chromatin-visualized with antibodies against histone H4. Furthermore, localization of Ime4 to sites of transcription is RNA-dependent, as staining for Ime4, but not for RNA Pol II, was reduced in an RNase-dependent manner (Haussmann, 2016).

Although m⁶A levels after G are low in Drosophila compared to other eukaryotes, broad co-localization of Ime4 to sites of transcription suggests profound effects on the gene expression landscape. Indeed, differential gene expression analysis revealed 408 differentially expressed genes where 234 genes were significantly upregulated and 174 significantly downregulated in neuron-enriched head/thorax of adult Ime4^null females. Cataloguing these genes according to function reveals prominent effects on gene networks involved in metabolism, including reduced expression of 17 genes involved in oxidative phosphorylation. Notably, overexpression of the m⁶A mRNA demethylase FTO in mice leads to an imbalance in energy metabolism resulting in obesity (Haussmann, 2016).

Next, tests were performed to see whether either of the two substantially divergent YTH proteins, YT521-B and CG6422, decodes m⁶A marks in Sxl mRNA. When transiently transfected into male S2 cells, YT521-B localizes to the nucleus, whereas CG6422 is cytoplasmic. Nuclear YT521-B can switch Sxl alternative splicing to the female mode and also binds to the Sxl intron in S2 cells. In vitro binding assays with the YTH domain of YT521-B demonstrate increased binding of m⁶A-containing RNA. In vivo, YT521-B also localizes to the sites of transcription (Haussmann, 2016).

To further examine the role of YT521-B in decoding m⁶A Drosophila strain YT521-B^MI02006 was analyzed, where a transposon in the first intron disrupts YT521-B. This allele is also viable, and phenocopies the flightless phenotype and the female Sxl splicing defect of Ime4^null flies. Likewise, removal of maternal YT521-B together with zygotic heterozygosity for Sxl and YT521-B reduces female viability and results in sexual transformations such as male abdominal pigmentation. In addition, overexpression of YT521-B results in male lethality, which can be rescued by removal of Ime4, further reiterating the role of m⁶A in Sxl alternative splicing. Since YT521-B phenocopies Ime4 for Sxl splicing regulation, it is the main nuclear factor for decoding m⁶A present in the proximity of the Sxl-binding sites. YT521-B bound to m⁶A assists Sxl in repressing inclusion of the male-specific exon, thus providing robustness to this vital gene regulatory switch (Haussmann, 2016).

Nuclear localization of m⁶A methylosome components suggested a role for this 'fifth' nucleotide in alternative splicing regulation. The discovery of the requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing has important implications for understanding the fundamental biological function of this enigmatic mRNA modification. Its key role in providing robustness to Sxl alternative splicing to prevent ectopic dosage compensation and female lethality, together with localization of the core methylosome component Ime4 to sites of transcription, indicates that the m⁶A modification is part of an ancient, yet unexplored mechanism to adjust gene expression. Hence, the recently reported role of m⁶A methylosome components in human dosage compensation further support such a role and suggests that m⁶A-mediated adjustment of gene expression might be a key step to allow for the development of the diverse sex determination mechanisms found in nature (Haussmann, 2016).

DEVELOPMENTAL BIOLOGY

In situ hybridization with vir antisense probes gives an unambiguous signal and is detectable in all somatic cells, but not in pole cells, at the formation of blastoderm. Thereafter, vir mRNA remains expressed in all cells, although not at the same level, throughout embryogenesis. There appears to be a gradual decrease in signal intensity from early (gastrulation) toward late embryogenesis. This was also observed on Northern blots where RNA extracts from 0- to 3-hr-old embryos showed a stronger hybridization signal than RNA derived from 3- to 17-hr-old embryos. Because a sexually mixed population of embryos did not fall into two classes after in situ hybridization, it is concluded that male as well as female embryos ubiquitously express vir RNA at the same level. Furthermore, a single transcript of 6 kb is detected in RNA derived from male and female adults as well as in RNA isolated from embryos and larvae (Niessen, 2001).

In situ hybridizations show that vir mRNA is expressed from blastoderm stage on throughout embryonic development. vir RNA levels appear highest at the beginning of gastrulation and then gradually decrease with increasing age. Little or no vir RNA is detected before blastoderm. This suggests that the maternal contribution to vir transcripts in the zygote is small. This is surprising because has been shown (Schutt, 1998) that removal of vir from the female germline results in abortive embryonic development, and this effect could not be rescued by a paternally contributed wild-type copy in the zygote. Three alternative explanations must be considered: (1) the mother contributes only low levels of vir transcripts for embryonic development; (2) Vir protein, but not VIR mRNA, is deposited into the egg; (3) it is possible that the mother does not deposit any vir activity at all into the developing zygote, but instead maternal vir is required solely during oogenesis to build a functional oocyte and is not required in the early zygote itself (Niessen, 2001).

EFFECTS OF MUTATION

The gene virilizer is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir^2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. These data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA (Hilfiker, 1995).

All the tested alleles that cause lethality to males and females are also deficient for the female-specific functions of dosage compensation and sex determination. These pathways are controlled by Sxl, and vir could thus be a regulator of Sxl. The role of Sxl is well established in the pathway of somatic sex determination. Its protein, besides regulating the splicing of its own pre-mRNA (autoregulation), promotes the female-specific splicing of the tra pre-mRNA (transregulation) by blocking the 3' splice site that is used in males. The most obvious and best understood aspect of vir is its role in sex determination, as demonstrated by XX;vir mutant cells forming male structures in genetic mosaics. The temperature-sensitive intersexual phenotype of X/X;vir^1ts/vir^1ts animals is rescued by the construct hs-tra-female that constitutively expresses the female mode of tra (Hilfiker, 1991). This indicates that vir acts upstream of tra. The new findings confirm the high hierarchical position of vir in the cascade of sex-determining genes and suggest that it plays a role in the regulation of Sxl and tra (Hilfiker, 1995).

These genetic arguments receive support from molecular analyses that show that surviving XX; vir^2f mle pseudomales produce mRNAs of Sxl and tra that are male-specifically spliced. The male-specific products of tra could be the consequence of the male-specific expression of Sxl. The next experiments, however, indicate that vir is also directly engaged in the splicing of tra transcripts (Hilfiker, 1995).

Since Sxl^M/+;vir^2f animals are efficiently rescued from the female lethal effect of vir^2f, but remain sexually transformed pseudomales or strongly masculinized intersexes, it is concluded that the sex-transforming effect of vir mutations is not exerted via Sxl alone. When the female-specific function of Sxl is constitutively provided in XX;vir^2f animals by Sxl^M or SxlcF1#19, the female-specific function of tra is still not guaranteed, but in addition appears to require an active product of vir. To test this conclusion the rescued animals were subjected to Western analysis. Substantial levels of Sxl protein are present in strong intersexes and pseudomales. Even animals with normal levels of Sxl, such as the intersexes, still splice the pre-mRNA of their tra gene largely in the male mode, as indicated by their strongly masculinized appearance. These intersexes are not mosaics of male and female cells, but display an intermediate sexual phenotype at the cellular level characteristic of flies expressing only low levels of female-specific products of tra. The results thus support the conclusion that vir is needed not only for female-specific expression of Sxl, but also of tra. This may also be the case for fl(2)d, although to a lesser extent, since XX animals mutant for fl(2)d are rescued by Sxl^M1 and are sexually transformed into intersexes at 29°C (Hilfiker, 1995).

The Western data furthermore suggest that presence of Sxl protein still requires vir for the splicing of its own pre-mRNA: pseudomales have substantially less Sxl protein than females which suggests that the transcripts produced by the Sxl plus allele in these genotypes are efficiently spliced only in the presence of vir plus. Based on these results, vir belongs to the class of genes that are involved in the regulation of Sxl. Recessive mutations in the genes fl(2)d and snf have been shown to prevent female-specific splicing of Sxl pre-mRNA in XX animals; and, like vir^2f, the female-specific mutant effects of both genes are rescued by Sxl^M mutations (Hilfiker, 1995 and references therein).

The sex-specific lethality of XX animals mutant for vir^2f by itself is suggestive of a role for vir in dosage compensation. Interactions between vir^2f and mutations in genes known to play a role in dosage compensation, such as Sxl and the male-specific lethal genes (msl genes) further support the proposed role of vir. XX animals with otherwise viable combinations of vir alleles, e.g. vir^1ts/vir^2f, die when they have only a single dose of Sxl plus. However, animals with two X chromosomes that are mutant for vir^2f are rescued by constitutive expression of Sxl. These results indicate that vir mutations achieve their female-specific lethal effect via Sxl, which explains why males are not affected by these same allelic combinations (Hilfiker, 1995).

Animals with two X chromosomes and mutant for vir^2f are also rescued, although with low frequency, by loss of function of msl genes. In view of the sex-transforming effect of mutations in vir, and since mutations in msl genes do not interfere with sexual differentiation, it is not surprising that the rescued animals are transformed into sterile pseudo-males. It is concluded that vir^2f allows the inappropriate activity of the msl genes in XX zygotes, and that the affected animals then die as a result of overexpression of X-linked genes. Elimination of any one of the msl genes lowers X-chromosomal transcription towards a level that is lethal for XY animals, but is appropriate for a single X in XX animals. However, the fact that the rescue of XX;vir^2f animals by mutations in msl genes is weak, is a warning that the regulatory network of dosage compensation is more complex. Sxl controls an early female-specific vital function that is not dependent on msl gene activity (Hilfiker, 1995 and references therein).

This interpretation is supported by the recent finding that the Mle and Msl-1 proteins bind to the polytene X-chromosomes in salivary glands and Malpighian tubules of XX animals mutant for vir^2f or vir^1ts; furthermore, these animals have the male-specifically acetylated form H4Ac16 of histone H4. Binding of the Mle and Msl-1 proteins to the X-chromosome and acetylation of histone H4 at lysine 16 normally occur only in males and are strongly, although not absolutely, correlated with hypertranscription of their single X-chromosome (Hilfiker, 1995).

How could vir participate in the regulation of the male specific lethal (msl) genes? Recent studies have shown that, of all the msl genes [mle; msl-1 and msl-3, only msl-2 is differentially expressed due to sex-specific splicing -- only males are capable of producing a functional protein; in females, the productive splice seems to be prevented by Sxl. Thus, it appears that the Sxl protein acts again, as in the regulation of its own mRNA and that of tra, by blocking a splice site in the msl-2 transcripts. Since the rescue of XX;vir^2f animals by Sxl^M is not complete and since the Sxl^M1/Y males survive despite the presence of Sxl, it is concluded that the Sxl product requires vir function to efficiently prevent the male-specific processing of msl-2 transcripts (Hilfiker, 1995).

It was unexpected that XX;vir^2f animals rescued by Sxl^M were transformed into pseudomales or strongly masculinized intersexes. A simple model in which vir acts above Sxl predicts that the rescued animals, due to the female-determining effect of Sxl, would be females; alternatively, if vir acted below Sxl, there would be no rescue of the lethality. The actual results are best interpreted by a model in which vir acts at both levels, upstream and downstream of Sxl, but ostensibly with differential effectiveness. Its function appears to be absolutely required for female-specific splicing of Sxl transcripts, but seems less important for the regulation of tra, and even less for msl-2 which is assumed to be the other target. This is inferred from the observation that the function provided by Sxl^M1 or Sxl^M4 in XX;vir^2f animals is largely, but not completely, sufficient to prevent the inappropriate activity of the msl genes, but insufficient to make enough female-specific products of tra necessary for female sexual development (Hilfiker, 1995).

vir^2f rescues XY males from the male-specific lethal effect of Sxl^M1 and partially of SxlcF1#19, but not of Sxl^M4. These results can be understood by recalling that Sxl^M1, in contrast to Sxl^M4, is not unconditionally constitutive, and XY animals mutant for Sxl^M1 and snf¹⁶²¹ or fl(2)d¹ can survive as males, some of which are even fertile. Surviving males of genotype Sxl^M1/Y;vir^2f do produce some Sxl protein, but not enough to make vir function dispensable. This implies that Sxl^M1, in addition to the functions of snf plus and fl(2)d plus, also requires vir plus to become fully functional in XY animals, and hence that an active product of vir must also be present in males (Hilfiker, 1995).

If vir is active in males, as these results imply, then it is unlikely to play a discriminatory role in sex determination. Rather, it may be comparable to da and tra2, two genes whose products are present in both sexes, but serve sex-determining functions only in females (Hilfiker, 1995).

Most alleles of vir are zygotic lethals. Since the first screen, many more vir alleles have been generated, and still only two, vir^1ts and vir^2f, are truly female-specific. This indicates that most lesions induced in vir affect a general non sex-specific function which, however, is dispensable in somatic cells of genetic mosaics: homozygous mutant cells survive in clones and in transplanted imaginal discs (Hilfiker, 1995).

Female-specific alleles and regular lethal alleles are also known for snf and fl(2)d, two genes that are, like vir, involved in the female-specific splicing of Sxl. Whereas putative null alleles of vir and fl(2)d are viable in cell clones, snf null is required for normal cell proliferation. The gene snf codes for the Drosophila homolog of the mammalian U1A snRNP, a general component of the splicing machinery. The vital function of vir, fl(2)d and snf, however, cannot depend on Sxl as this gene is completely dispensable in males, and constitutive expression of Sxl does not rescue animals mutant for those alleles that abolish this vital function (Hilfiker, 1995).

If, as the results suggest, vir is involved in splicing of the pre-mRNAs of Sxl and tra, and perhaps msl-2, it could also participate in the splicing of pre-mRNAs of vital genes. Mutations in snf, in fl(2)d, and in vir, can cause lethality to both sexes. However, since clones of vir and fl(2)d, in contrast to those of snf null, are viable in males and females, neither vir nor fl(2)d can encode a general and absolutely necessary factor for cell growth and cell viability. It is not known whether vir is directly involved in the splicing of tra and msl-2 transcripts, or whether it somehow activates the Sxl protein (Hilfiker, 1995).

The continuous production of Sxl proteins in XX animals is maintained by autoregulation and depends on virilizer. In the soma, clones of XX cells lacking either Sxl or vir are sexually transformed and form male structures; in the germline, XX cells mutant for Sxl extensively proliferate, but are unable to differentiate. The role of vir in the germline has been studied by generating germline chimeras. XX germ cells mutant for vir, in contrast to cells mutant for Sxl, perform oogenesis. The early production of Sxl in undifferentiated germ cells is independent of vir; later, in oogenesis, expression of Sxl becomes dependent on vir. It is concluded that the early Sxl proteins are sufficient for the production of eggs whereas the later Sxl proteins are dispensable for this process. However, vir must be active in the female germline to allow normal embryonic development because maternal products of vir are required for the early post-transcriptional regulation of Sxl in XX embryos and for a vital process in embryos of both sexes (Schutt, 1998).

daughterless (da), hermaphrodite (her) and snf are known to have a maternal effect on the viability of daughters. XX embryos from da or her mutant mothers fail to initiate Sxl. vir is a further component that is maternally required for the regulation of Sxl in XX embryos. Unlike da and her, but similar to snf, it acts at the level of post-transcriptional control. The establishment promoter of Sxl is activated both in vir⁶/plus and vir^2f/vir^2f embryos derived from vir mutant mothers, but Sxl expression is not maintained. The lethality of vir⁶/plus embryos indicates that maternal product of vir is responsible for the autoregulation of Sxl during early development. In addition, maternal vir function is also required for viability of offspring of both sexes as shown by strong alleles disrupting the vital function. For such alleles, neither the autoregulation of Sxl nor the vital process can be rescued by a paternal vir plus allele. The reason may be that the zygotic gene is either not yet active or not yet sufficiently active (Schutt, 1998).

Most genes of the somatic sex determination cascade are dispensable within germ cells. Sxl, although necessary for oogenesis, does not have a master regulatory function for sex determination in the germline, as indicated by the following observations. (1) Expression of Sxl in germ cells is first detected in 16- to 20-hour-old embryos. A male-specifically expressed gene, however, is already observed in germ cells of 10-hour-old embryos, suggesting that some aspect of sexual development must have been determined prior to expression of Sxl. (2) Sxl^f4/Sxl^f1 and Sxl^f5/Sxl^f1 larvae have female gonads by the criteria of size and morphology; only as adults do the germ cells form abnormal multicellular cysts. Sxl does not seem to control the sex-specific differentiation of germ cells in larvae, but is required later for oogenesis. (3) XY germ cells containing Sxl^M1 or Sxl^M4 are not feminized, but instead form fertile sperm (Schutt, 1998 and references therein).

The expression of Sxl in the germline depends on inductive signals from the gonadal soma and on an autonomous signal given by the germline X:A ratio, which is measured by elements different from those used to determine the somatic X:A ratio. Thus, initiation of Sxl is different in soma and germline. In a second phase, expression of Sxl in differentiating cysts becomes dependent on vir. This parallels the regulation of Sxl in somatic cells. In contrast to the soma, however, the consequences of misexpression of Sxl are different: germ cells are not sexually transformed (Schutt, 1998).

The different germline phenotypes of Sxl and vir mutations may be due to trivial reasons. A perdurance effect of Vir protein made in heterozygous stem cells prior to mitotic recombination could maintain the production of Sxl protein in vir clones. However, the tested females continue to lay eggs even two weeks after the induction of clones and after several rounds of cell divisions. By this time, Vir protein should have been diluted or eliminated and should no longer be able to regulate Sxl. A second possibility would be that the first 24 hours of development in heterozygous condition before the induction of clones determines the female fate of a germ cell irreversibly. In all previous experiments with ovaries mutant for Sxl^f4 and with transplanted pole cells mutant for Sxl^f1, the germ cells were homozygous mutant from the beginning. But even when Sxl clones were induced under the same conditions as the vir clones, the females remained sterile as in earlier experiments showing that Sxl is necessary after the induction of the clones. Therefore, the phenotypic differences of Sxl and vir mutants are caused by different requirements for the two genes (Schutt, 1998).

Based on these results, the following model is proposed. The initiation mechanism of Sxl in the germline is unknown. However, for female development, early Sxl protein is necessary in gonial cells at the tip of the germarium in the adult ovary. Later in oogenesis, the germ cells may become independent of the primary signals by the soma and the X:A ratio and maintain Sxl production by autoregulation. This later post-transcriptional regulation depends on vir. Late Sxl protein, however, is no longer necessary for the female differentiation of germ cells, once these cells have embarked on the oogenic pathway. It is not yet clear what other functions Sxl has in later stages of oogenesis. Sxl protein is redistributed during oogenesis. In stem cells and early cystoblasts in the germarium, Sxl is predominantly cytoplasmic. During the mitotic divisions, the level of cytoplasmic Sxl drops drastically. In the cluster of 16 cells, the protein becomes concentrated in the nuclei of the cystocytes. This transition of Sxl protein may reflect the early and late functions of Sxl. It is concluded that Sxl is required for a short period during the transition from stem cells to cystoblasts. Lack of Sxl during this phase results in tumorous cysts (Schutt, 1998).

snf and fl(2)d are two other genes involved in the autoregulation of Sxl in the soma. These two genes are also required for oogenesis. Mutant females produce no eggs, but instead form multicellular cysts. It can only be speculated why mutations in snf, fl(2)d and vir have different consequences in the germline. They may still all affect the autoregulation of Sxl, as they do in somatic cells, but do so at different times. It is possible that the production of early Sxl protein depends on a post-transcriptional regulation that requires snf and fl(2)d, whereas vir may only act later in oogenesis (Schutt, 1998).

The regulation of Sxl in the germline is certainly more complex than in the soma, and the germline function of this gene is also less clear. A comparative analysis of the regulation and function of Sxl in soma and germline is therefore worthwhile and may contribute to an understanding of how a gene operates in different cell types (Schutt, 1998).

Initial analysis of >30 vir alleles shows that most of them represent lesions undetectable by whole genome Southern analysis. Two exceptions are the alleles vir²² and vir²³, which are both associated with small deletions of genomic DNA. Another 5 alleles have also been detected at this level of analysis, but show rather large deletions of >30 kb each. To identify potentially relevant domains, the lesions were determined in the two female-specific alleles, vir^1ts and vir^2f, and in three strong alleles, vir⁴, vir²², and vir²³, which are recessive lethals for males and females. Genomic DNA was isolated from adult flies homozygous for the sex-specific and viable vir^1ts or vir^2f alleles and from third instar larvae, hemizygous for the lethal alleles vir⁴, vir²², or vir²³ over Df(2R)vir130 (Hilfiker, 1995). vir²³ is a 202-bp deletion, and vir²² is a deletion of 295 bp associated with an insertion of 2 bp. Both introduce frameshifts in the ORF so that the wild-type sequence is interrupted. The proteins produced by these two alleles contain 339 and 1261 amino acids of the wild-type conceptual translation product, respectively, followed by a short stretch of amino acids until the next STOP codon. The third lethal allele, vir⁴, is a single base exchange that leads to a nonsense mutation at position 1177 in the deduced amino acid sequence. All three lethal alleles are hence predicted to produce truncated proteins of differnt lengths. The two sex-specific allels, vir^1ts and vir^2f, are point mutations that lead to single amino acid exchanges. In vir^1ts, glutamic acid at position 1423 is mutated into lysine (1423 E-K), and in vir^2f, methionine 1283 is replaced by lysine (1283 M-K). The latter two point mutations lie relatively close together and could define a region in Vir that is required primarily for correct functioning in Sxl autoregulation (Niessen, 2001).

The products of Sxl, tra, and tra-2 are known regulators of alternative splicing decisions in Drosophila but they are not essential for processes other than sex determination (and dosage compensation, in the case of Sxl) because males that are null for these genes are viable and appear phenotypically normal. However, additional genes [fl(2)d, virilizer, and l(2)49Db] are required for correct control of alternative splicing decisions by Sxl are also essential for viability in both sexes; hence, their products may also have roles in other alternative splicing events. To determine whether these include the control of Ubx alternative splicing, it was asked whether the Ubx isoform ratios are altered in heterozygotes for mutations in these genes. In contrast to the stability described in the preceding section, the Ubx splicing pattern is altered significantly when the expression or function of virilizer or fl(2)d is reduced. The strongest effect is observed with virilizer, using a loss-of-function allele (vir³) that is recessive lethal in both sexes. In heterozygous larvae the proportion of Ubx class I mRNAs declines while that of classes II and IV increases. The proportion of class I that contains the B element is not altered. The increase in classes II and IV indicates that inclusion of both mI and mII is reduced but that the effect on mI exceeds that on mII. Inclusion of mI is also reduced in adults, although the effect was weaker than in larvae (Burnette, 1999).

REFERENCES

Search PubMed for articles about Drosophila virilizer

Burnette, J. M., Hatton, A. R. and Lopez, A. J. (1999). Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs ofmDrosophila melanogaster. Genetics 151: 1517-1529. 10101174

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