Sex lethal


Table of contents

Regulation of splicing by SXL: 1. General considerations

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer, by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains (R1 and R2) and an 80 amino acid C-terminus. The functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions have been investigated. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains; proteins that consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. These results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. It is speculated that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. The interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain (Samuels, 1998).

Sex-lethal (Sxl) is an RNA-binding protein containing two conserved RNA binding domains (RBDs) and a glycine-rich region that functions as a regulator of alternative splicing in Drosophila sex determination. Sxl monomers interact cooperatively upon binding to target RNAs; this cooperativity depends on the glycine-rich N terminus. Band shift experiments were used to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog). Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis. Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, are suggested by the yeast two-hybrid assay. In a systematic examination of the RNA binding properties of Sxl domains, the Sxl termini as well as the RBDs influence RNA binding specificity. Selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure (Wang, 1997).

The Drosophila Sex-lethal protein is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within SXL and Transformer (TRA) pre-mRNAs. In an examination of SXL mRNA binding specificity, SXL prefers polyuridine stretches surrounded by purine residues. SXL appears to recognize and preferentially bind to a polyuridine stretch with a downstream AG sequence (Sakashita, 1994).

Sex lethal has two transcripts, early and late, with different promoters and dramatically different splicing patterns. The Sxl early transcripts are activated transiently in early embryos by a female-specific promoter and have a unique 5' exon (E1) located between late exons 1 and 2. Exon E1 is spliced to exon 4, which is common to all SXL transcripts, skipping both exons 2 and 3 (Keyes, 1992). In contrast, the late SXL transcripts derive from an essentially constitutive promoter but are spliced sex specifically. The male-specific exon, exon 3, is included by default in all male transcripts and contains in-frame nonsense codons that block SXL protein production. In the presence of SXL protein, the late transcripts skip exon 3 and splice in the female pattern. The function of the SXL early transcripts, which are present only briefly, is to generate an initial pulse of SXL protein that serves to direct splicing of the late SXL transcript into the female mode. After the initial phase, SXL female splicing and female development are maintained throughout life by SXL protein generated from the late transcripts in a positive autoregulatory loop. It is essential that both early and late SXL splicing be correctly regulated, because inappropriate levels of SXL expression in either males or females will lead to lethality arising from inappropriate X-chromosome dosage compensation. Even though the early transcripts are present briefly in early embryogenesis whereas the late transcripts are present virtually throughout life, no embryo-specific splicing factors are needed for the early splice. Neither are sex-specific factors required. Instead, the early splicing pattern is dependent on whether the 5' splice site region originates from exon E1 or exon 2 (Zhu, 1997).

As part of a cascade of genes that are regulated by sex-specific splicing, SXL controls the sex-specific splicing of Transformer (TRA) RNA and also its own RNA. SXL contains two RNA-binding domains and is known to bind TRA pre-mRNA near the alternative 3' splice site, thus blocking use of that site to give the female-specific splicing pattern. SXL not only binds SXL pre-mRNA near the alternative 3' splice site but also at distant, multiple sites surrounding the SXL alternative exon. Moreover, SXL binds cooperatively at these multiple sites. The SXL amino terminus is essential for the cooperative interaction and is also required for regulatory activity in vivo. It appears that this region of SXL protein, which resembles regions in some other RNA-binding proteins, is a domain that mediates protein-protein interactions during RNA binding and plays an important role in splicing regulation (Wang, 1994).

The RNA binding activity of SXL was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Both RNA binding domains (RBDs) are required in cis for site-specific RNA binding. Individual RBDs interact with RNA more weakly and lose the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts (Kanaar, 1995).

The mechanism for generating female Transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. In addition, a blockage mechanism is involved in Sex-lethal autoregulation. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. The splice signals at the exon are suboptimal with regard to alternate splicing (Horabin, 1993a).

Sex lethal antagonizes the general splicing factor U2AF65 to regulate splicing of TRA. Transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 were used to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on TRA splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain TRA splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of SXL pre-mRNA. These results are a direct proof that SXL, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing (Granadino, 1997).

Splicing mechanism of SXL: 2. Sxl's role in Transformer pre-mRNA splicing

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site (Sosnowski, 1989).

Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal, is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. This study addresses the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. Co-transfection experiments were performed in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, it was found that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing (Inoue, 1990).

The transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to transformer do not show regulation. Mutations which replace the 16 nucleotides surrounding the branch point or alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations which reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors (Sosnowski, 1994).

The Sex-lethal (Sxl) protein regulates alternative splicing of the Transformer (TRA) messenger RNA precursor by binding to the tra polypyrimidine tract during the process of sex-determination. Sxl binds tightly to a characteristic uridine-rich polypyrimidine tract at the non-sex-specific 3' splice site in one of the TRA introns, preventing the general splicing factor U2AF from binding to this site and forcing it to bind to the female specific 3' splice site. The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The Sxl-binding polypyrimidine tract of TRA-mRNA does not form a tertiary base-paired structure. The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers an insight into how a protein binds specifically to a cognate RNA without that RNA possessing any intramolecular base-pairing (Handa, 1999).

The Drosophila protein Sex-lethal (Sxl) contains two RNP consensus-type RNA-binding domains (RBDs) separated by a short linker sequence. Both domains are essential for high-affinity binding to the single-stranded polypyrimidine tract (PPT) within the regulated 3' splice site of the transformer (tra) pre-mRNA. In this paper, the effect of RNA binding to a protein fragment containing both RBDs from Sxl (Sxl-RBD1 + 2) has been characterized by heteronuclear NMR. Nearly complete (85%-90%) backbone resonance assignments have been obtained for unbound and RNA-bound states of Sxl-RBD1 + 2. A comparison of amide 1H and 15N chemical shifts between free and bound states has highlighted residues that respond to RNA binding. The beta-sheets in both RBDs (RBD1 and RBD2) form an RNA interaction surface, as has been observed in other RBDs. A significant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sxl have different or nonanalogous points of interaction with the tra PPT. R142 (in RBD2) exhibits the largest chemical shift change upon RNA binding. The role of R142 in RNA binding was tested by measuring the Kd of a mutant of Sxl-RBD1 + 2 in which R142 was replaced by alanine. This mutant lost the ability to bind RNA, showing a correlation with the chemical shift difference data. The RNA-binding affinities of two other mutants, F146A and T138I, were also shown to correlate with the NMR observations (Lee, 1997).

Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior

The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). This study shows instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. The existence of this unknown Sxl target was inferred from the observation that a constitutively feminizing tra transgene that restores fertility to tra- females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra-. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, this pathway is referred to as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, it was found that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila (Evans, 2013).

Developmental regulatory pathways are rarely as simple as they first appear, but as the twist to the Drosophila sex-determination pathway this study reports here suggests, complications can provide clues to evolution. It was shown that Sxl, the rapidly evolved target of the Drosophila primary sex-determination signal, no longer can be regarded as transmitting all its feminizing orders other than size dimorphism to the soma exclusively through its well-known switch-gene target tra. Instead, one must distinguish between a major pathway branch, TSF, in which tra is sufficient to dictate feminization, and a minor branch, TIF, in which it is not (Evans, 2013).

Evidence for the TIF branch derives from female-viable but masculinizing combinations of partial-loss-of-function Sxl alleles that fail to induce either TSF or TIF in diplo-X individuals, so that when TSF-branch activity is restored by constitutively feminizing transgene U2af-traF or, even more definitively, by a constitutively feminizing mutant endogenous tra allele, mutant females remain TIF defective and hence sterile. Although TIF-mutant sterility superficially resembles sterility in TSF-mutant transgenics, in that both phenotypes include a failure to lay eggs, the TIF-mutant block to egg laying occurs at ovulation, whereas that in TSF-defective transgenics occurs later at oviposition. The possibility that the kind of branch in the TIF pathway that is reported in this study might exist was suggested first in a previous paper reporting the behavior of some U2af-traF-feminized gynandromorphs (coarse-grained XX//XO mosaics) in which the failure of Sxl to activate what is now known to be the TIF pathway was a consequence of the absence of a female primary sex-determination signal in TRA-F-feminized Sxl+ XO cells (Evans, 2007) rather than a consequence of Sxl mutations in TRA-F–feminized XX cells. Because 38% of the feminized egg-producing gynandromorphs failed to lay their eggs, it is concluded that there must be some functionally Sxl- XO somatic cells that cannot substitute for the XX somatic cells required for egg laying, even when feminized by TRA-F. Although gynandromorphs are not nearly as convenient as Sxl-mutant females for studying TIF, they do strengthen the argument that TIF-defective sterility is not caused either by a upset in dosage compensation or by some idiosyncrasy of U2af-traF in Sxl-mutant females (Evans, 2013).

Strong evidence is necessary to legitimize the TIF claim because of the surprising finding that SXL-F functioning in the TIF pathway takes place in a subset of neurons that sex-specifically express fru mRNAs. Because fru sex-specific splicing is controlled entirely by TRA-F, the simplest model would suggest that any deficiency in the sex-specific functioning of these neurons reflects a TSF defect. Of course, just because fru is sex-specifically regulated in these neurons does not require that fru be solely or even partially responsible for their feminization in every case (Evans, 2013).

At this point the 'I' in TIF necessarily stands for 'insufficient' rather than 'independent.' Because conditions under which the TIF phenotype was studied were all ones in which TRA-F activity for the TSF pathway was provided at a level sufficient to rescue the sterility of tra− females, no evidence for or against independence could be generated. If, as the fru neuron results might suggest, tra works with one or more unknown Sxl targets to achieve full feminization in some neurons, the name ultimately might have to be changed to something like 'tra-partnered feminization.' Discovering the identity of the Sxl TIF-gene targets and the specific neurons in which they are required would provide the tools necessary to resolve this question about the relationship between TSF and TIF. The recent availability of an enormous panel of well-characterized neuronal GAL4 drivers should be a great help in this connection, particularly in view of the finding that GAL4-driven SXL-F expression can rescue the TIF-mutant phenotype in females while causing little damage to males. The gene female-specific-independent-of-transformer seemed to be a promising candidate TIF-pathway target until it was shown that, contrary to a previous study, it is firmly in the TSF pathway (and hence is in need of renaming) (Evans, 2013).

The ovulation block should be particularly amenable to future genetic and developmental analyses designed to identify targets of the TIF because it is particularly suited to positive genetic selection in a suppression screen. Arguing for the potential of such a suppression screen is the fact mentioned above that fertility could be restored to TIF-defective females by a GAL4 driver/ SXL-F target combination that had relatively little adverse effect on male viability or fertility. Such sex specificity suggests that the set of neurons responsible for the TIF ovulation defect may not be very large and that disruption of their normal controls is unlikely to disrupt non–sex-specific aspects of development (Evans, 2013).

This report introduces several genetic tools, among which the GAL4 target UAS-Sxlalt5-C8 is perhaps the most broadly useful. That this transgene, which conditionally generates both exon-5 alternative SXL-F isoforms, provides relatively strong Sxl+ function while having no adverse effect on females indicates that the adverse effect on females caused by the Sxl GAL4 target previously reported, a transgene that encodes only a single exon-5 isoform, may not reflect a normal activity of SXL-F protein. Another useful tool is Dp(1;1)SxlΔPm, which can expand the utility of various partial-loss-of-function Sxl alleles. This tool is a chromosomal duplication of Sxl truncated at its 5′ end so that it lacks the gene's maintenance promoter but retains an intact establishment promoter and all the activities that transiently active promoter elicits. The response of this truncated Sxl allele to the female X-chromosome dose signal, a response that ends during the early blastoderm stage, can facilitate engagement of the Sxl positive-feedback loop for various Sxl-mutant alleles without otherwise influencing their Sxl-mutant phenotype. For example, Dp(1;1)SxlΔPm is particularly useful in combination with the intriguing double mutant Sxlf18,f32 because together they can generate thoroughly masculinized Sxl-mutant adult females (pseudomales) with far higher viability and longevity than any previously described masculinizing Sxl genotype. Last, two dominant temperature-sensitive lethal balancers that were introduced in this study should be generally useful, because they allow crosses to be designed so that daughters with one combination of a maternal and paternal X chromosome of choice are the only progeny to survive (Evans, 2013).

Sex determination for flies in the family Drosophilidae is unlike that for most other higher insects in many fundamental respects, including having Sxl rather than tra as the target of their primary sex-determination signal and having Sxl rather than tra as the gene whose positive-feedback loop on its own pre-mRNA splicing maintains the female developmental pathway commitment. Although the TIF branch could be a recent addition to the Drosophila sex-determination pathway made well after Sxl had taken over tra's role as the master feminizing gene, a more intriguing possibility is that TIF instead may reflect an ancestral function that Sxl acquired in the earliest step on its evolutionary path toward usurping tra's role as master sex switch. Because both TIF and TSF function in neurons that sex-specifically express fru, perhaps the first female- specific function that Sxl acquired was to modify the developmental functioning of fru in some neurons. Initially this function may have been achieved without the need for a sex-specific Sxl product, with sex-specific products coming only later as fine-tuning of that particular function under the control of tra. The switch from tra as a regulator of Sxl to Sxl as a regulator of tra (a switch that could have been facilitated by the development of redundancy in the positive-feedback circuits for the two genes) would make any female-specific gene target of Sxl that existed before the switch be independent of tra regulation today if its control by Sxl persisted. Of course there are many important questions about the remarkable path taken by Sxl functional evolution and the forces that drove those changes for which an understanding of the TIF pathway might not be relevant. How did Sxl come to respond to an X-chromosome dose signal? How did it come to control X-chromosome dosage compensation? Why is Sxl's control of germ-line sex determination so different from its control of sex determination in the soma? On the other hand, because next to nothing is known about any of these questions, it is hard to predict where clues might lead regarding an early female-specific Sxl function that the TIF pathway might help reveal. Regardless of whether the TIF pathway is ancestral or recent, further analysis leading to the discovery of the SXL-F targets in this regulatory branch undoubtedly will advance understanding how genes control behavior and how SXL-F proteins control RNA functioning (Evans, 2013).

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. This study addresses the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. This dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior (Chau, 2012).

Sxl has a pivotal role in the cell fate switch from a self-renewing GSC to a differentiation-competent CB. This study addressed the underlying cellular mechanism by identifying nanos as a Sxl target gene. nanos is a conserved translational repressor that is thought to maintain GSC fate by silencing a set of as-yet-unidentified differentiation-promoting mRNAs. Cell fate switching occurs as one of the daughter cells initiates a differentiation program that includes significant accumulation of the Bam protein and rapid down-regulation of a set of GSC specific markers, including Nanos protein. Previous studies have shown that Nanos protein expression is dynamically regulated at the level of translation such that Nanos protein levels are high in GSCs but undetectable in all Bam-expressing daughter cells. This study found that Sxl is responsible for this dynamic expression pattern. Moreover. regulation is direct. nanos RNA is bound by the Sxl protein in ovarian extracts, and nanos silencing is dependent on the presence of Sxl binding sites located in the nanos 3' UTR. Together these studies point to a mechanism in which Sxl promotes the GSC/CB cell switch by lowering Nanos protein levels. The use of a female-specific factor to control nanos expression is consistent with earlier studies showing that the mechanism regulating GSC differentiation differs significantly between the sexes; for example, neither bam nor nanos is required for this process in males, although they are expressed. Thus, the Sxl-mediated posttranscriptional regulatory mechanism described in this study provides a direct link between sexual identity and execution of the appropriate differentiation pathway (Chau, 2012).

Sxl is expressed in both GSCs and their progeny, raising the question of how its role in Nanos down-regulation is restricted to Bam-expressing cells. bam itself may confer cell-type specificity, as it too is required for lowering Nanos protein levels, although there are, as yet, no physical data to support direct regulation. Nevertheless, taken together with the genetic studies that reveal that bam requires Sxl activity to promote differentiation, these observations suggest that cell-type specificity could be achieved by a regulatory complex containing Sxl and Bam. Both Sxl and Bam have been shown to repress translation in other contexts, further suggesting that the two proteins might function together to directly repress nanos translation. Biochemical studies will be required to test this model (Chau, 2012).

It was previously shown that Sxl is required for germ cells to progress from a stem cell to a differentiation-competent CB fate, and that if this pathway is blocked, the mutant germ cells form a tumor. Although this study shows that inappropriate nanos expression is necessary for tumor growth, genetic epistasis experiments indicate that nanos expression is not responsible for malignant transformation because the majority of surviving double-mutant germ cells continue to resemble a tumor cell. This conclusion is supported by studies showing that forced expression of nanos in GSC progeny is not sufficient to block differentiation. Even in a genetically sensitized background where an accumulation of extra stem cell–like germ cells has been observed, germ cells proceed through oogenesis. Therefore, these studies suggest that although forced expression of nanos is responsible for expanding the number of mutant germ cells, aberrant regulation of other genes and/or pathways under Sxl control elicit malignant transformation. The identification and analysis of additional Sxl targets genes will offer insights into how the failure to successfully exit the stem cell state is connected to the genesis of germ cell tumors (Chau, 2012).

Table of contents

Sex lethal: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D. BR>

The Interactive Fly resides on the
Society for Developmental Biology's Web server.