sans fille

DEVELOPMENTAL BIOLOGY

Embryonic

In contrast a nuclear distribution during most of development, antibody staining of early embryos (0-30 minutes of development) reveals that SNF is non-nuclear. Several mitotic division later, SNF is restricted to the nuclei (Flickinger, 1994).

Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. The evolution of this machinery was studied using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the Drosophila contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. These data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction (Kroiss, 2008).

Splicing of pre-mRNAs is catalyzed by the spliceosome, a macromolecular machine consisting of a large number of protein factors and the uridine-rich small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6, and U5. The biogenesis of these particles occurs in a stepwise manner. First, nuclear-transcribed, m⁷G-capped snRNAs U1, U2, U4, and U5 are exported into the cytoplasm, where a conserved sequence motif in these RNAs (Sm site) serves as a binding platform for the seven Sm proteins B/B', D1, D2, D3, E, F, and G. As a consequence, a ring-shaped Sm core domain is formed. This domain is crucial for subsequent steps in the biogenesis of UsnRNPs, such as formation of the hypermethylated m^2,2,7G cap and import of the assembled particle into the nucleus. At a yet to be defined step, additional factors are recruited to form the mature UsnRNP particles that function in splicing (Will, 2001; Kroiss, 2008 and references therein).

Previous studies have shown that Sm proteins bind spontaneously, albeit in a hierarchical manner, onto UsnRNAs in vitro). However, in cellular extracts, this process depends on ATP and the activity of the multisubunit SMN complex. Recently, a systematic interaction study on the human SMN complex has established its basic architecture. A modular composition was deduced where the three factors SMN, Gemin2, and Gemin8 form the backbone of the entire complex. Onto this core, the peripheral building blocks Gemin3/4 and Gemin6/7/UNRIP bind to form the functional unit. In support of this modular architecture, Gemin-containing subcomplexes have been identified composed of SMN/Gemin2, Gemin3-Gemin5, and Gemin6/7/UNRIP (Kroiss, 2008 and references therein)

The SMN complex not only functions in the assembly of the Sm core domain, but also influences additional steps in the biogenesis pathway of UsnRNPs. One such step is the nuclear import of the assembled UsnRNP, which is mediated by the SMN complex (or parts thereof) in conjunction with the import factor importin β. In addition, specific UsnRNP proteins and the cap hypermethylase Tgs1 have been found in association of the SMN complex (Mouaikel, 2003). This observation indicates that the SMN complex coordinates various events during UsnRNP biogenesis by assuming the role of a binding platform for the respective assisting factors (Kroiss, 2008).

The multisubunit composition of the human SMN complex has impeded the mechanistic dissection of the UsnRNP assembly process. Thus, although RNA interference studies indicated essential roles of several Gemins in the assembly reaction, their precise contributions remain unclear. To facilitate mechanistic studies and to gain insight into the evolution of the SMN complex, genomic databases were mined for organisms that lack individual Gemins and hence may contain a simpler assembly machinery. Indeed, this was the case for different organisms including dipterans. Drosophila was chosen for further investigation because of the wealth of genetic resources. An affinity chromatography strategy has permitted the purification of an assembly-active complex composed of SMN and Gemin2 only. Remarkably, this complex not only facilitated assembly of the Sm core domain but also discriminates between cognate and noncognate RNAs. Thus, a combined bioinformatic and biochemical approach revealed that the assembly reaction requires only two core proteins in vitro, even though SMN complexes from most metazoans are of considerable complexity. It is speculated that Gemins 3-8 have been recruited to the SMN complex in the course of evolution to integrate assembly with additional steps in the biogenesis of UsnRNPs (Kroiss, 2008).

To understand how the UsnRNP assembly machinery has evolved, homology searches were performed for all Gemin proteins constituting the human SMN complex in genomic databases of a variety of organisms. Because of its diverse functions and its transient cytoplasmic interaction with the SMN complex, the UNRIP protein has been excluded from this analysis (Kroiss, 2008).

SMN and Gemin2 orthologs (termed Yab8p and Yip1p) but no other Gemins can be found in the fungus Schizosaccharomyces pombe. Importantly, both orthologs interact physically and may hence form a functional unit. In Saccharomyces cerevisiae, however, only the distantly related Gemin2 ortholog Brr1p, but no SMN ortholog, could be identified. Brr1p has been proposed to be an ortholog of human Gemin2. However, because of its limited homology to Yip1p this finding has been questioned. Taking advantage of the dramatically increased genome databases and novel search algorithms (PSI BLAST), Brr1p can be defined as the single significant homolog of human Gemin2 and S. pombe Yip1p. Because reciprocal searches further support this homology, Brr1p is the ortholog of human Gemin2. Thus, S. cerevisiae retained only one Gemin and hence is unlikely to form a functional SMN complex. Interestingly, like S. pombe, the plants Arabidopsis thaliana and Oryza sativa contained orthologs of SMN and Gemin2 only. It is therefore concluded that SMN and Gemin2 represent the most primitive and ancestral version of the SMN complex. Surprisingly, the genome of Dictyostelium discoideum, a facultative multicellular organism, encoded orthologs of Gemin3 and Gemin5. Given that D. discoideum is basal to fungi and metazoans, the bioinformatic data suggest that both Gemins have been lost during evolution in the fungi branch but were retained in metazoans. Interestingly, the presence of a Gemin5 ortholog in Ostreococcus tauri but its absence in land plants indicates an independent gene loss in this phylum. Moreover, it was found that SMN and Gemins 2, 3, 5, 6, 7, and 8 are present in the cnidarian Nematostella vectensis, a basic metazoan. Gemin4 first appeared in the sea urchin Strongylocentrotus purpuratus, whereas it is absent in all ecdysozoans under study. This suggests that Gemin4 has joined the SMN complex only recently in evolution, most likely with the appearance of deuterostomians. Consequently, it was found to be part of the SMN complex in vertebrates such as Danio rerio but also cephalochordates like Branchiostoma floridae and Ciona intestinalis (urochordates). Thus, plants and some fungi possess a core complex composed of SMN and Gemin2 only, whereas an elaborate SMN complex has developed only in animal branches by addition of Gemin proteins (Kroiss, 2008).

The bioinformatic data indicated an evolutionary trend in the animal kingdom toward a multisubunit SMN complex (see Evolution of an RNP assembly system). Interestingly, however, no orthologs of most Gemins were found in the dipterans Drosophila melanogaster and Anopheles gambiae although they were present in closely related Apis mellifera and Nasonia vitripennis. Further analysis was restricted to D. melanogaster in this study. Besides the known SMN ortholog (Miguel-Aliaga, 2000; Rajendra, 2008), a Gemin2 ortholog was found encoded by CG10419 and putative orthologs of Gemin3 (Dhh1) and Gemin5 (Rigor mortis). Dhh1 protein shows high conservation in the N-terminal DEAD box helicase domain but possesses a diverged C terminus. Rigor mortis displays moderate homology to Gemin5 over the entire protein length. A phylogenetic analysis revealed that both evolve significantly faster than their orthologs in other organisms. This released evolutionary pressure might indicate the emergence of a novel function or the loss of a common one for these factors. These data suggest that D. melanogaster possesses a much simpler SMN complex as compared with vertebrates (Kroiss, 2008).

To investigate whether Dhh1 and Rigor mortis have retained their function in the context of the D. melanogaster SMN (dSMN) complex, use was made of a novel epitope tag. This tag consists of the first 30 aa of human SMN protein, which are specifically recognized by the monoclonal antibody 7B10 (Meister, 2000). Importantly, competition with synthetic peptide comprising this epitope allows native elution of tagged proteins from this antibody. Because D. melanogaster SMN protein lacks these 30 aa, a plasmid was constructed allowing the expression and subsequent purification of a protein fused to this epitope (termed TagIt epitope) after stable transfection of Schneider2 cells. In a TagIt-Dhh1 affinity purification, only small amounts of dSMN protein could be detected under physiological conditions but not at salt concentrations exceeding 250 mM. Thus, Dhh1 is only weakly associated with dSMN. Similarly, the role of Rigor mortis was investigated. No binding of Rigor mortis to dSMN has been observed, arguing against a stable association of this protein with the dSMN complex. These data suggest that Rigor mortis either functions in UsnRNP core formation in a manner different from vertebrate Gemin5 or has completely lost its function in the pathway of UsnRNP biogenesis (Kroiss, 2008).

To gain detailed insight into the composition of the D. melanogaster SMN complex, a TagIt-dSMN-expressing Schneider2 cell line was generated. Importantly, TagIt-dSMN was incorporated into a high-molecular-weight complex that also contained Gemin2. This implied that the tagged dSMN protein engages in interactions similar to those of its endogenous counterpart. The SMN complex was affinity-purified from extracts by means of 7B10 affinity chromatography. Affinity-purified proteins were separated by SDS-PAGE under reducing and nonreducing conditions and identified by protein mass spectrometry and Western blotting. Whereas the tagged SMN protein and its interactor dGemin2 could be readily identified, neither Dhh1 nor Rigor mortis was found under the purification conditions applied in this study (Kroiss, 2008).

It is known that the human SMN complex consists of the core machinery (i.e., SMN and Gemins) as well as the transiently interacting substrates that are transferred onto the UsnRNA during assembly. These are the Sm proteins and some UsnRNP-specific proteins. Strikingly, the entire set of Sm proteins, namely SmB, SmD1 (gene snRNP69D), SmD2 (CG1249), SmD3, SmE (CG18591), SmF (DebB), and SmG (CG9742), was prominently present in the elution. Furthermore, the UsnRNP-specific factors U1 70KU2A', the U2B''/U1A ortholog SNF, and the ortholog of the U5 specific protein (CG4849) U5 116kD were found reproducibly in the purified complex. However, the abundance of these specific proteins varied among preparations and was often substoichiometric (Kroiss, 2008).

During UsnRNP assembly, the SMN complex physically contacts the UsnRNAs (Fischer, 1997). In vertebrates, this interaction has been proposed to be mediated, at least in part, by Gemin5 and to occur in the cytoplasm. Interestingly, despite the absence of Rigor mortis in the TagIt-dSMN complex, snRNAs U1, U2, U4, and U5 were specifically coprecipitated with dSMN and dGemin2 antibodies from total Schneider2 cell extract. Identical results were obtained when the SMN complex was purified from the cytosol, where SMN is predominantly localized. Hence, in D. melanogaster, the SMN complex is sufficient to recruit a set of substrate proteins similar to those in vertebrates. In addition, the complex interacts specifically with U snRNAs in the cytoplasm, which reflects a situation previously observed in Xenopus laevis oocytes (Kroiss, 2008).

Previous studies have indicated that Gemins interact within the SMN complex in a modular manner. Interestingly, homology searches for components of the SMN complex in a variety of organisms have recapitulated this finding on an evolutionary scale. The most simple SMN-containing complex is composed of SMN and Gemin2 only and can be found in unicellular organisms such as the fission yeast S. pombe and in plants. The next level of complexity is characterized by the appearance of Gemin3 in D. discoideum, thus predating the emergence of the Fungi/Metazoa clade. The absence of Gemin5 from genomes of fungi and land plants and its presence in the green algae O. tauri and in D. discoideum indicate independent secondary gene loss in fungi and plants. This may be due to a role of Gemin5 outside of the SMN complex, which is not retained in these organisms (Kroiss, 2008).

Only later in evolution at the level when first metazoans developed, the building block composed of Gemins 6, 7, and 8 was added to the set of the Gemin family. From this time on, organisms had the potential to express an SMN complex similar in architecture to the human one. The only component that was not present at that point was Gemin4, which can be found only in the genomes of deuterostomians. Thus, the data suggest that the SMN complex evolved by a blockwise addition of Gemins to an ancient core complex of SMN and Gemin2 in a manner corresponding to their mutual biochemical association (Kroiss, 2008).

In striking contrast to this overall evolutionary trend, a remarkable simplification of this complex in the dipterans was found A. gambiae and D. melanogaster. In these animals, no orthologs of Gemin4 were found, as expected, but also of Gemins 6-8 were absent. However, these latter Gemins were clearly present in hymenopterans. Although orthologs of Gemin3 and Gemin5 were found in dipterans, they show a significantly higher evolutionary rate in dipterans than in other clades. These computational findings have been experimentally challenged by a biochemical approach that has allowed isolation of an assembly-active SMN complex from D. melanogaster. Indeed, the composition of the complex was remarkably simple and consisted of SMN and Gemin2 as the only stoichiometric components. Dhh1 (Gemin3) bound to this core complex only at low salt concentrations, and Rigor mortis (Gemin5) was not present at all. The D. melanogaster SMN complex therefore equals its counterpart in S. pombe and plants although the function of SMN and Gemin2 orthologs in these organisms has not been demonstrated. It is conceivable that Dhh1 and Rigor mortis have adopted novel functions in a different context because they rapidly diverge from their ancestors. Consistent with this notion, a function of Rigor mortis (Gates, 2004) in ecdysone signaling has been described (Kroiss, 2008).

Despite the obvious simplicity of the SMN complex in D. melanogaster, this study has provided evidence that this unit is functionally related to the SMN complex of mammals. First, a set of UsnRNP-related substrates, namely the common Sm proteins, UsnRNP specific factors (U1 70K, U2A', U2B''/U1A, and U5 115K), and UsnRNAs were found to be part of the complex. Most of these factors have previously been shown to bind to SMN complexes of vertebrates (Meister, 2002). Second, affinity-purified dSMN complex mediate the assembly of the Sm core domain in vitro. Similar to the situation in humans, a strong dependence was found of UsnRNP core assembly on temperature but not on ATP (Meister, 2002). However, at present the possiblility cannot be ruled out that assembly of UsnRNPs in D. melanogaster cytosolic extracts requires ATP hydrolysis as observed for the same reaction in vertebrates (Kroiss, 2008).

The obvious simplicity of the assembly system of D. melanogaster allowed the reconstitution of the dSMN complex from recombinant proteins and the investigation of its mode of action. Interestingly, strong cooperativity was observed in Sm protein binding onto the complex. Heterooligomers D1/D2 and B/D3 had only little affinity for the complex, but binding was greatly enhanced in the presence of recombinant Sm heterooligomer E/F/G. Further studies are required to determine the precise binding sites of all Sm proteins on the SMN complex and to test the influence of arginine methylation on Sm protein binding. It was an open question why UsnRNP assembly is strictly dependent on the SMN complex in vivo even though this reaction is spontaneous in vitro. Assembly studies with the D. melanogaster SMN complex show that precise assembly of the Sm core domain on UsnRNA was possible only when Sm proteins were prebound to the SMN complex, whereas misassembly of isolated Sm proteins occurred under the same conditions. In addition, human SMN and Gemin2 are likewise sufficient to specifically transfer Sm proteins onto UsnRNA. Hence, these data and similar studies performed in vertebrates argue for a dual role of the SMN complex as an RNP assembler and chaperone (Kroiss, 2008).

From an evolutionary point of view, these findings raise the question why dipterans can afford a minimized assembly system, whereas apparently other branches in the animal kingdom require a multicomponent SMN complex. The most plausible explanation for this paradox is that Gemins 3-8 are not primarily involved in the assembly reaction per se but rather in other steps during the UsnRNP biogenesis. Thus, it is known that the human SMN complex integrates several steps in biogenesis, such as cap hypermethylation and nuclear import. It is speculated that these steps will occur in dipterans independent of the SMN complex and may hence allow for the omission of individual Gemins. Further studies will be needed to test whether this is indeed the case (Kroiss, 2008).

In conclusion, these studies have shown that the integration of bioinformatics and biochemistry can be used to analyze cellular pathways functionally and evolutionarily. Similar strategies may prove to be powerful tools in the analysis of even more complex systems such as the spliceosome (Kroiss, 2008).

Adult

All nuclei in both the germ-line and the somatic components of the ovary are NSF protein positive. High levels are found in the cytoplasm of nurse cells, suggesting that the SNF protein is deposited into the developing oocyte when the nurse cells dump their contents (Flickinger, 1994)

Effects of mutation or deletion

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, a comparison has been carried out between the sex determination mechanism that operates in the germline and that which operates in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL mRNA is maintained by a positive autoregulatory feedback loop in which Sxl protein ensures its continued synthesis by binding to SXL pre-mRNA and thereby imposing the productive (female) splicing mode. Ectopic expression of Sxl protein triggers the female-specific Sxl mRNA feedback loop in male germ cells without disrupting spermatogenesis. There is no adverse effect on male viability or fertility. The presence of Sxl protein may sometimes retard the rate of differentiation of spermatocytes, but does not abort the process (Hager, 1997).

The gene splicing-necessary factor (sans fille or snf), which encodes a component of U1 and U2 snRNPs, participates in SXL RNA splicing control. An increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. Female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. In fact, increased doses of snf+ and Sxl+ can feminize germ cells when germ cells have an altered X chromosome to autosome ratio. snf+ and Sxl+ feminize 2X3A germ cells. Flies with the higher doses of snf+ display a greater proportion of yolky germline cysts and eggs. Somatic sex is important in this feminization as the sexual phenotype of all internal and external somatic dimorphic characters appears to be fully female in 2X3A animals carrying a heat-shock transformer transgene. What then is the role of Snf in the germ-line? It seems likely that Snf acts to boost the autoregulatory effectiveness of very low levels of female Sxl protein, rather than acting directly on its own to influence SXL transcript splicing. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu, which control female-specific processes in the ovary (Hager, 1997).

Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate, either forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability (Oliver, 1988). snf's role in germline sex determination was determined directly from the genetic analysis of a single allele, snf1621. Females homozygous for snf1621 are sterile. In the mutant neither the oocyte nor the nurse cells differentiate. Instead the germinal cells continue to divide, resulting in the formation of ovarian tumors. A similar phenotype is seen in fused, ovo, and otu mutants. The similarity of snf and Sxl mutant phenotypes in the germline suggests that they disrupt the same process: germline sex determination (Salz, 1992 and references).

In contrast to snf's role in germline sex determination, its role in somatic sex differentiation and X chromosome dosage compensation can only be inferred by an unusual genetic interaction between mutations at the snf and Sxl loci. Although doubly heterozygous females have reduced viability, many surviving females show signs of sex transformations. snf is required for the activation of Sex-lethal in both the germline and the soma (Salz, 1992).

To determine unequivocally the null phenotype of snf, deletions were prepared of the entire snf-coding sequence. Animals homozygous for such a deletion suffer embryonic lethality (Flickinger, 1994).

In Drosophila, females require products of the gene Sxl for sex determination, dosage compensation and fertility. The X-chromosomal gene snf, located in 4F1 to 4F11 and previously called fs(1)1621, provides maternal and zygotic functions necessary for Sxl activity in germ line and soma. In XX animals, the mutation SxlM1 which was reported to express the female-specific functions of Sxl constitutively can rescue all phenotypes resulting from lack of snf product. XY animals carrying SxlM1 and lacking maternal or zygotic snf activity survive as males with some female traits. A stock was constructed in which the females are snf SxlM1/snf SxlM1 and males snf SxlM1/Y. This shows that SxlM1 is not truly expressed constitutively in animals with an X:A ratio of 0.5, but requires activity of snf for initiation or maintenance (Steinmann-Zwicky, 1988).

The two sexes of Drosophila melanogaster are distinguished by a two-fold difference in the dose of a small set of specific X-linked genes -- the so-called numerator elements -- which collectively determine the transcriptional state of the switch gene Sex-lethal (Sxl). During a 45-minute window of time very early in development the numerator elements do this through their actions on the Sxl 'establishment' promoter, Sxl_Pe. The double dose of numerator elements in chromosomal females (XX) triggers transcription at Sxl_Pe whereas the single dose in chromosomal males (XY) leaves this promoter off. However, a very different mechanism then operates to maintain the functional state of Sxl. This maintenance process exhibits Sxl gene dosage effects with levels snf⁺ gene product. Thus, although Sxl interacts with a variety of RNAs to control a diversity of functions, only the autoregulatory aspect of Sxl is affected by increased Snf. Encoded by sans fille, Snf is the Drosophila homolog of mammalian U1A and U2B" and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that must be physically separate from Snf's functioning within snRNPs. This observation adds to evidence that the functional relationship between these two genes is very different from that between Sxl and other genes that affect Sxl pre-mRNA splicing (Cline, 1999)

Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, Snf has been shown to be rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either the maternal or zygotic snf dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a⁶⁰, has no effect. Increased snf⁺ enhances Sxl autoregulation even when U1-70k and SF3a⁶⁰ are reduced by mutation to levels that, in the case of SF3a⁶⁰, demonstrably interfere with Sxl autoregulation. The observation that increased snf does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a⁶⁰ is additional evidence that snf dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function (Cline, 1999).

From the effects of raising the dose of the wild-type snf gene above normal levels, it is inferred that the integral snRNP protein encoded by snf acts outside of the snRNP in controlling pre-mRNA splicing for Sxl. One would not pick snf as a gene likely to display phenotypic effects of increased dose because snf encodes only one of many proteins that make up U1 and U2 snRNPs. In the genetically sensitized system used here to reveal snf⁺ dose effects, these complex multimeric assemblies are at levels that suffice for all of the needs of the organism. Such dose effects are not typical of integral snRNP proteins because increasing the dose of the gene encoding the U1 protein U1-70k or that encoding the U2 protein SF3a⁶⁰ has no effect on Sxl autoregulation. This negative result is particularly meaningful in light of the demonstration that while lowering the level of SF3a⁶⁰ interferes with Sxl autoregulation, this does not eliminate the effects of increased snf⁺ dose (Cline, 1999).

Could the influence of increased snf⁺ dose reflect a quirk of fruit fly regulatory circuitry in which snRNP levels are tied to U1A/U2B" levels? A priori, this would seem a disadvantageous strategy for the fly to use. Because most RNA splicing involves a sensitive balance between competing potential splice sites that one might expect to be affected by changes in the levels of these two snRNPs, one would expect regulatory circuitry to insulate the general splicing system from perturbation, not tie it to a single gene product in this way. Moreover, because a maternal effect of increased snf⁺ dose is observed that is nearly as striking as the zygotic dose effect, such a sensitive regulatory connection would have to operate both maternally during oogenesis to govern subsequent snRNP levels in the embryo and zygotically to govern snRNP levels at later stages. Two experimental observations argue against such a tie to snf. (1) Although striking effects on Sxl by even a single extra copy of snf⁺ are seen in various sensitized situations, males and females wild-type for Sxl can carry as many as 10 extra copies of the same snf⁺ construct and be fully viable. (2) Most damaging for this unlikely hypothesis, increasing snf⁺ dose does not suppress the mutant phenotypes caused by decreasing the level of U1-70k or SF3a⁶⁰ (Cline, 1999).

In contrast, if Snf functions specifically in Sxl autoregulation not as an integral component of U1 or U2 snRNPs but as an individual protein, the snf⁺ dose effects would not be reflecting changes in functional snRNP levels, but simply the established tendency of metazoan gene product levels to be roughly proportional to structural gene dose. Dose effects in this case would be indicating Snf's key participation in the process by which Sxl protein inhibits the male Sxl pre-mRNA splice by binding to RNA, a process likely to directly involve relatively few proteins (Cline, 1999).

The fly's use of U1A/U2B'' as an alternative splicing factor in sex determination would not be the first case of an integral spliceosomal protein acting outside of the snRNPs. Non-snRNP mammalian U1A negatively regulates its level by binding to sites in U1A pre-mRNA to block polyadenylation. U1A may also function more generally to couple splicing and 3' end formation. Such pleiotropy raises the possibility of an undiscovered world of biological functions for integral snRNP proteins operating as free agents. Because these proteins also have essential housekeeping functions, their other roles might not be easily revealed in vivo. Positive autoregulation gives the Sxl assay used here an extremely nonlinear character that surely facilitated study of biochemical effects that might otherwise have been too small to detect (Cline, 1999).

How might Snf be involved in Sxl autoregulation? There is evidence that a small fraction of Snf is in proximity to Sxl on RNA. Previous models have assumed that any interaction between Snf and Sxl occur with Snf acting as part of U1 or U2 snRNPs; it is suggested that this interaction is preceded by Sxl binding to pre-mRNA between exons 3 and 4 to block the male splice. Through an interaction between Snf within the snRNPs and Sxl bound to RNA surrounding the male exon, an abortive presplicing complex for exon-3 has been proposed to form, allowing the alternative exon 2-4 female-specific splice to proceed by default (Cline, 1999 and references therein).

In light of the data reported here, it now appears that Snf may bind with Sxl to pre-mRNA flanking the male exon, perhaps each facilitating or stabilizing the other's binding. By this model, it would not be surprising if the consequences of such an association were most significant at low concentrations of Sxl, such as those which surely prevail in the sensitized situations describe here. In addition to stabilizing Sxl binding, or even as an alternative to it, non-snRNP Snf associating with Sxl may be necessary to inhibit further spliceosomal complex assembly around the male-specific exon 3. Perhaps independent Snf protein interacting with Sxl bound to the pre-mRNA interferes with an essential association that Snf in the snRNPs themselves would need to have with other splicing factors to define exon 3 splice sites (Cline, 1999).

The dose-sensitive involvement of snf in somatic Sxl autoregulation described here is one of the strongest similarities between the regulation of sex-specific gene expression in the soma and in the germ line. It was shown earlier that simply increasing the dose of snf⁺ in an otherwise wild-type fly can trigger female-specific splicing of Sxl transcripts in male germ cells. For the soma, increasing snf⁺ alone will not suffice to engage the autoregulatory splicing loop; however, somatic Sxl regulation can be made nearly as sensitive to increased snf⁺ dose as germline Sxl regulation by alleles such as Sxl^Mf1 that are so weak that by themselves they do not lower male viability or fertility. The ease with which Sxl splicing control in the soma can be made to respond to the dose of RNA splicing factors favors the idea that the ancestral system controlling the sex-specific expression of Sxl in both the germline and the soma might have been based entirely on dose effects of RNA splicing factors (Cline, 1999).

In view of the central and remarkably specific role snf plays in controlling sex-specific expression of Sxl, it is a curious coincidence that the only genus known to use Sxl as a master sex switch is also the only genus with a species known to use a single protein, Snf, for tasks that two proteins, U1A and U2B'', handle in species as diverse as potatoes and humans. Learning how closely the evolution of Sxl as the master sex-determination gene for Drosophila was paralleled by the evolution of this difference in integral U1 and U2 snRNP proteins might suggest what the driving forces were that led to both changes (Cline, 1999).

The Drosophila U1-70K protein is required for viability, but its arginine-rich domain is dispensable

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain's importance in intact multicellular organisms have not been carried out. This paper reports a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, it was found that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, it was shown that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together these studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function (Salz, 2004).

A striking outcome of this study is the finding that U1-70K can accomplish its vital function in the absence of an RS domain. The failure to detect a phenotype in these mutant animals challenges the prevailing view that the U1-70K RS motif provides a vital link between splicing regulators and the U1 snRNP. It is suggested instead that either the interactions detected in vitro are not essential in vivo or there are multiple means by which the U1 snRNP can interact with splicing regulators. Support for the latter view comes from the demonstration that U1 snRNP particles lacking both the U1-70K RS domain and SNF can no longer support viability. Synthetic lethality is attributable to the simultaneous loss of two functions that contribute to the same activity or pathway. Interestingly, in S. cerevisiae synthetic lethal interactions have been observed between nonlethal mutations in several different U1 snRNP components. Thus, the in vivo results argue that while disruption of the U1-70K RS-mediated protein links has no detectable consequence to the living organism, the simultaneous disruption of multiple connections causes U1 snRNP function to fall below the level needed to support development and viability (Salz, 2004).

In Drosophila, the female-specific Sex-lethal (Sxl) protein is required for oogenesis, but how Sxl interfaces with the genetic circuitry controlling oogenesis remains unknown. An allele of sans fille (snf) that specifically eliminates Sxl protein in germ cells was used to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype. It was found that tumor growth requires both Cyclin B and zero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by wild-type germ cells. Using a series of molecular markers, it was established that while the tumor often contains at least one apparently bona fide germline stem cell, the majority of cells exhibit an intermediate fate between a stem cell and its daughter cell fated to differentiate. In addition, snf tumors misexpress a select group of testis-enriched markers, which, remarkably, are also misexpressed in ovarian tumors that arise from the loss of bag of marbles (bam). Results of genetic epistasis experiments further reveal that bam's differentiation-promoting function depends on Sxl. Together these data demonstrate a novel role for Sxl in the lineage progression from stem cell to committed daughter cell and suggest a model in which Sxl partners with bam to facilitate this transition (Chau, 2009).

The observation that female germ cells lacking Sxl are tumorigenic was first published >20 years ago, yet the place of this female-specific RNA binding protein in the genetic circuitry controlling oogenesis has remained elusive. This study investigated Sxl's role in the germline by taking advantage of a snf mutant allele that specifically eliminates Sxl expression in the germline. Genetic and cell biological analysis established that Sxl is required for the transition from stem cell to committed daughter cell by showing that the majority of Sxl-deficient germ cells have acquired an intermediate fate. These findings are in contrast to the commonly held view, based on fusome morphology alone, that Sxl mutant germ cells arrest development later in the differentiation pathway. This study also offers new insight into the function of bam by demonstrating that its differentiation-promoting function depends on Sxl and, importantly, that Sxl and bam control the same sex-specific expression network (Chau, 2009).

In current models, maintenance of GSC identity requires contact with the niche to trigger the signal transduction cascade required for transcriptional repression of bam. This in turn provides a permissive environment that allows PUM, which forms a complex with its partner protein Nanos (NOS), to inhibit translation of a yet unidentified set of mRNAs required for differentiation. Differentiation begins when one of the daughter cells is displaced from the niche and can no longer receive the signals that silence bam transcription. Bam then initiates the differentiation program by antagonizing the translation-inhibitory functions of the PUM/NOS complex. This model predicts a strong negative correlation between the expression of bam and the GSC markers, and, while this is true in general, there have been reports of rare single cells that coexpress bam and one or more GSC-specific markers. These and other studies have suggested that cells fated to differentiate first pass through an intermediate stage that transitions, without dividing, to a mature cystoblast (Chau, 2009).

It was shown that Sxl is required to complete the transition from GSC to a mature cystoblast (CB) by demonstrating that the majority of germ cells lacking Sxl resemble an immature CB-like cell. Furthermore, genetic epistasis experiments suggest that the failure to progress beyond this intermediate stage is attributable to a lack of bam function. This conclusion is supported by studies showing that the tumors resulting from the lack of Sxl and bam are remarkably similar. Specifically, the loss of Sxl and bam results in germ cell tumors with the same unique molecular signature including expression of stem cell markers and with the same set of testis-enriched markers. Both types of germ cell tumors also require CycB and zpg for growth. This comparison reveals that snf and bam tumors both result from a failure to initiate the differentiation pathway in stem cell progeny. It will be interesting to determine what role the misregulated testis-enriched markers play in this process (Chau, 2009).

On the basis of these data, it is proposed that Sxl partners with bam to facilitate the transition between GSCs and the daughter cell that is fated to differentiate. In females, differentiation via control of bam transcription is initiated in response to position-dependent extrinsic cues from the somatic gonad. Extrinsic cues from the somatic gonad also provide essential sex-specific information, via control of Sxl expression. These findings suggest that the intrinsic Sxl/bam partnership serves to integrate these two different extrinsic signaling pathways. This proposal is particularly compelling because it explains how bam function is substantially different in males and females (Chau, 2009).

How might Sxl and bam function converge to promote female germ cell differentiation? Sxl acts post-transcriptionally to repress splicing and translation. The molecular function of Bam, on the other hand, is unknown but is also thought to act post-transcriptionally. At a genetic level, one function of bam is to antagonize the differentiation-inhibiting activity of PUM/NOS. The presence of putative high-affinity Sxl-binding sites in both the 5'-UTR and the 3'-UTR of the nos mRNA leads to the speculation that Sxl functions with Bam to promote differentiation by inhibiting the translation of nos. Although this model is consistent with the finding that Sxl and Bam are coexpressed in the appropriate cell type, biochemical studies to address this point have proved to be technically challenging (Chau, 2009).

In summary, these studies support a model in which the Sxl/bam pathway is required for germ cells to progress from a stem cell fate to a differentiation-competent CB fate. These studies also suggest that if this pathway is blocked, germ cells will continue to proliferate, forming a tumor. It is proposed that the block in the developmental progression from stem cell to fully committed daughter cell is the initial tumorigenic event. This model is consistent with the general view that adult stem cells are the source of some, and perhaps all, tumors. Not only do some human germ cell tumors display many of the same characteristics as the Drosophila tumors described in this study, including expression of stem cell markers, but also they occur frequently in individuals with intersex disorders. While true orthologs of Sxl and bam are not found in vertebrates, the processes that they regulate are likely to be conserved. Future studies aimed at understanding the functional connections between the failure to engage the Sxl/bam genetic programs, misexpression of testis-enriched markers, and tumorigenesis will likely provide mechanistic insight into the pathogenesis of germ cell tumors in humans (Chau, 2009).

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