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).

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).

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