Sex lethal


Table of contents

Splicing mechanism of SXL: 3. Proteins involved in splicing of Sxl mRNA

The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. P-element-mediated mutagenesis has been used to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. (1) The P-element insertion that inactivates fl(2)d interrupts this ORF. (2) Amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. (3) All of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts (Penalva, 2000).

How can Fl(2)d affect the function of Sxl? One possibility is that Fl(2)d plays an important role in post-translational modifications or proper subcellular localization of Sxl. A second possibility is that Fl(2)d has a direct role in the regulation of pre-mRNA splicing. It could act, for example, by facilitating Sxl binding to its target pre-mRNAs or by assisting its repressive activities. These putative functions could be based on direct interactions between Sxl and Fl(2)d. Alternatively, Fl(2)d could be part of a complex in which Sxl functions, which could also include the products of the genes snf and vir. Finally, a third possibility is that Fl(2)d facilitates the use of the distal splice sites in Sxl and tra that become activated when Sxl represses the use of the proximal ones. More indirect effects of Fl(2)d, as is likely to be the case for the recently reported effects on Sxl expression of mutations in an aspartyl-tRNA synthetase gene, cannot be ruled out (Penalva, 2000).

The non-sex-specific function of fl(2)d remains to be identified. Because Sxl activity is not required for male development, mutations that affect both male and female viability cannot be attributed to genetic interactions with Sxl. It is also very unlikely that Fl(2)d is a general splicing factor involved in an obligatory step in the splicing reaction for the following reasons: (1) no aberrant splicing pattern is detected in Sxl and tra RNAs in fl(2)d mutant males or females (the normal, default sites are used); (2) fl(2)d mutations are not cell lethal. Particularly interesting are the results of the clonal analysis of fl(2)d2, a mutation that produces a truncated, presumably nonfunctional protein. Clones homozygous for fl(2)d2 induced in fl(2)d2/+ males and females are viable, except that in females they develop male instead of female structures, due to the female-specific function of fl(2)d. Furthermore, transplanted male germ cells homozygous for fl(2)d2 can develop into functional spermatozoa, whereas transplanted female germ cells homozygous for fl(2)d2 follow an abortive spermatogenetic pathway, which is an indication of a sexual transformation of the mutant germ cells, due to the female-specific function of fl(2)d. If Fl(2)d was a component of the general splicing machinery, neither fl(2)d2 homozygous clones nor mutant germ cells would survive. One possible scenario is that Fl(2)d plays a role in splicing regulation of a gene(s) important for development in addition to those involved in sex determination. Recently, it has been reported that fl(2)d appears to be necessary for inclusion of mI and mII microexons in Ubx mRNAs. Other examples of splicing factors that are essential for viability but that are dispensable for processing of multiple pre-mRNAs have been described in yeast, Drosophila, and mammals. For some of these, mutations have been identified that disrupt the splicing of only particular substrates, similar to the effects of the fl(2)d1 mutation (Penalva, 2000).

The gene virilizer is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. These data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA (Hilfiker, 1995).

All the tested alleles that cause lethality to males and females are also deficient for the female-specific functions of dosage compensation and sex determination. These pathways are controlled by Sxl, and vir could thus be a regulator of Sxl. The role of Sxl is well established in the pathway of somatic sex determination. Its protein, besides regulating the splicing of its own pre-mRNA (autoregulation), promotes the female-specific splicing of the tra pre-mRNA (transregulation) by blocking the 3' splice site that is used in males. The most obvious and best understood aspect of vir is its role in sex determination, as demonstrated by XX;vir mutant cells forming male structures in genetic mosaics. The temperature-sensitive intersexual phenotype of X/X;vir1ts/vir1ts animals is rescued by the construct hs-tra-female that constitutively expresses the female mode of tra. This indicates that vir acts upstream of tra. The new findings confirm the high hierarchical position of vir in the cascade of sex-determining genes and suggest that it plays a role in the regulation of Sxl and tra (Hilfiker, 1995).

These genetic arguments receive support from molecular analyses that show that surviving XX; vir2f mle pseudomales produce mRNAs of Sxl and tra that are male-specifically spliced. The male-specific products of tra could be the consequence of the male-specific expression of Sxl. The next experiments, however, indicate that vir is also directly engaged in the splicing of tra transcripts (Hilfiker, 1995).

Since SxlM/+;vir2f animals are efficiently rescued from the female lethal effect of vir2f, but remain sexually transformed pseudomales or strongly masculinized intersexes, it is concluded that the sex-transforming effect of vir mutations is not exerted via Sxl alone. When the female-specific function of Sxl is constitutively provided in XX;vir2f animals by SxlM or SxlcF1#19, the female-specific function of tra is still not guaranteed, but in addition appears to require an active product of vir. To test this conclusion the rescued animals were subjected to Western analysis. Substantial levels of Sxl protein are present in strong intersexes and pseudomales. Even animals with normal levels of Sxl, such as the intersexes, still splice the pre-mRNA of their tra gene largely in the male mode, as indicated by their strongly masculinized appearance. These intersexes are not mosaics of male and female cells, but display an intermediate sexual phenotype at the cellular level characteristic of flies expressing only low levels of female-specific products of tra. The results thus support the conclusion that vir is needed not only for female-specific expression of Sxl, but also of tra. This may also be the case for fl(2)d, although to a lesser extent, since XX animals mutant for fl(2)d are rescued by SxlM1 and are sexually transformed into intersexes at 29°C (Hilfiker, 1995).

The Western data furthermore suggest that presence of Sxl protein still requires vir for the splicing of its own pre-mRNA: pseudomales have substantially less Sxl protein than females which suggests that the transcripts produced by the Sxl plus allele in these genotypes are efficiently spliced only in the presence of vir plus. Based on these results, vir belongs to the class of genes that are involved in the regulation of Sxl. Recessive mutations in the genes fl(2)d and snf have been shown to prevent female-specific splicing of Sxl pre-mRNA in XX animals; and, like vir2f, the female-specific mutant effects of both genes are rescued by SxlM mutations (Hilfiker, 1995 and references therein).

The sex-specific lethality of XX animals mutant for vir2f by itself is suggestive of a role for vir in dosage compensation. Interactions between vir2f and mutations in genes known to play a role in dosage compensation, such as Sxl and the male-specific lethal genes (msl genes) further support the proposed role of vir. XX animals with otherwise viable combinations of vir alleles, e.g. vir1ts/vir2f, die when they have only a single dose of Sxl plus. However, animals with two X chromosomes that are mutant for vir2f are rescued by constitutive expression of Sxl. These results indicate that vir mutations achieve their female-specific lethal effect via Sxl, which explains why males are not affected by these same allelic combinations (Hilfiker, 1995).

Animals with two X chromosomes and mutant for vir2f are also rescued, although with low frequency, by loss of function of msl genes. In view of the sex-transforming effect of mutations in vir, and since mutations in msl genes do not interfere with sexual differentiation, it is not surprising that the rescued animals are transformed into sterile pseudo-males. It is concluded that vir2f allows the inappropriate activity of the msl genes in XX zygotes, and that the affected animals then die as a result of overexpression of X-linked genes. Elimination of any one of the msl genes lowers X-chromosomal transcription towards a level that is lethal for XY animals, but is appropriate for a single X in XX animals. However, the fact that the rescue of XX;vir2f animals by mutations in msl genes is weak, is a warning that the regulatory network of dosage compensation is more complex. Sxl controls an early female-specific vital function that is not dependent on msl gene activity (Hilfiker, 1995 and references therein).

How could vir participate in the regulation of the male specific lethal (msl) genes? Recent studies have shown that, of all the msl genes [mle; msl-1 and msl-3, only msl-2 is differentially expressed due to sex-specific splicing -- only males are capable of producing a functional protein; in females, the productive splice seems to be prevented by Sxl. Thus, it appears that the Sxl protein acts again, as in the regulation of its own mRNA and that of tra, by blocking a splice site in the msl-2 transcripts. Since the rescue of XX;vir2f animals by SxlM is not complete and since the SxlM1/Y males survive despite the presence of Sxl, it is concluded that the Sxl product requires vir function to efficiently prevent the male-specific processing of msl-2 transcripts (Hilfiker, 1995).

It was unexpected that XX;vir2f animals rescued by SxlM were transformed into pseudomales or strongly masculinized intersexes. A simple model in which vir acts above Sxl predicts that the rescued animals, due to the female-determining effect of Sxl, would be females; alternatively, if vir acted below Sxl, there would be no rescue of the lethality. The actual results are best interpreted by a model in which vir acts at both levels, upstream and downstream of Sxl, but ostensibly with differential effectiveness. Its function appears to be absolutely required for female-specific splicing of Sxl transcripts, but seems less important for the regulation of tra, and even less for msl-2 which is assumed to be the other target. This is inferred from the observation that the function provided by SxlM1 or SxlM4 in XX;vir2f animals is largely, but not completely, sufficient to prevent the inappropriate activity of the msl genes, but insufficient to make enough female-specific products of tra necessary for female sexual development (Hilfiker, 1995).

vir2f rescues XY males from the male-specific lethal effect of SxlM1 and partially of SxlcF1#19, but not of SxlM4. These results can be understood by recalling that SxlM1, in contrast to SxlM4, is not unconditionally constitutive, and XY animals mutant for SxlM1 and snf1621 or fl(2)d1 can survive as males, some of which are even fertile. Surviving males of genotype SxlM1/Y;vir2f do produce some Sxl protein, but not enough to make vir function dispensable. This implies that SxlM1, in addition to the functions of snf plus and fl(2)d plus, also requires vir plus to become fully functional in XY animals, and hence that an active product of vir must also be present in males (Hilfiker, 1995).

In Drosophila, the gene Sex-lethal is required for female development. It controls sexual differentiation in the soma, dosage compensation and oogenesis. The continuous production of SXL proteins in XX animals is maintained by autoregulation and depends on virilizer (vir). This gene is required in somatic cells for the female-specific splicing of Sxl primary transcripts and for an unknown vital process in both sexes. In the soma, clones of XX cells lacking Sxl or vir are sexually transformed and form male structures; in the germline, XX cells mutant for Sxl extensively proliferate, but are unable to differentiate. The role of vir has been studied in the germline by generating germline chimeras. XX germ cells mutant for vir, in contrast to cells mutant for Sxl, perform oogenesis. The early production of SXL in undifferentiated germ cells is independent of vir while, later in oogenesis, expression of Sxl becomes dependent on vir. It is concluded that the early SXL proteins are sufficient for the production of eggs whereas the later SXL proteins are dispensable for this process. However, vir must be active in the female germline to allow normal embryonic development because maternal products of vir are required for the early post-transcriptional regulation of Sxl in XX embryos and for a vital process in embryos of both sexes. Unlike daughterless and hermaphrodite, but similar to snf, Vir acts at the level of post-transcriptional control. The establishment promoter of Sxl is activated in vir mutant embryos but Sxl expression is not maintained. The lethality of vir6/+ embryos indicates that the maternal product of vir is responsible for the autoregulation of Sxl early in development. Maternal vir function is also required for viability of offspring of both sexes, as shown by strong alleles disrupting vitality. For such alleles, neither the autoregulation of Sxl nor the vital process can be rescued by a paternal vir+allele. the reason may be that the zygotic gene is either not yet active or not yet sufficiently active (Schutt, 1998).

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific Sex lethal RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is Sans-fille (Snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether Snf acts as part of an intact snRNP or a free protein. Evidence is provided that Sex lethal interacts with Sans-Fille in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with Sex lethal. Moreover, Sex lethal associates with other integral U1 snRNP components, and genetic evidence is provided to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link Sex lethal with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which Sex lethal represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, these studies are difficult to reconcile with the current model that proposes that the Sex lethal blocks splicing at the second catalytic step, and instead argue that the Sex lethal protein acts after splice site recognition, but before catalysis begins (Nagengast, 2003).

The Sxl male exon is unusual in that it contains two 3' AG dinucleotides separated by a short polypyrimidine tract. Interestingly, although the upstream 3' splice site is used almost exclusively for exon ligation in tissue-culture cells, both 3' splice sites are required for Sxl-mediated male-exon skipping. Moreover, crosslinking studies in HeLa cell extracts have shown that the U2AF heterodimer binds to the downstream 3' splice site and the intervening polypyrimidine tract, suggesting that U2AF may play an active role in Sxl regulation. These biochemical data have been validated by demonstrating that the Sxl protein can associate with the Drosophila U2AF orthologs. More importantly, genetic data provide compelling support for the biological relevance of these interactions by demonstrating that in females, the small subunit is important for both Sxl male-exon skipping and female viability. In addition to demonstrating a role for U2AF in Sxl autoregulation, this genetic result is notable because previous studies have failed to find RNA splicing defects associated with small subunit mutations. Whether this success reflects substrate-specificity or sensitivity of the assay remains to be determined (Nagengast, 2003).

In addition to controlling the use of the male exon 3' splice site, these studies suggest that Sxl controls the use of the male-exon 5' splice site by interacting with the U1 snRNP. This connection was established in three ways: (1) it was found that mutation of a single residue in the N-terminal RRM of SNF compromises both complex formation with Sxl and assembly into the U1 snRNP, thus suggesting that the two events are linked; (2) it has been demonstrated that, in addition to SNF, Sxl can associate with other integral U1 snRNP components, including the U1-70K protein and the U1 snRNA in whole cell extracts; (3) genetic interaction data provide evidence that U1-70K, like SNF, is important for the successful establishment of the Sxl autoregulatory splicing loop in females (Nagengast, 2003).

Although the discovery that SNF is an snRNP protein was the first clue that Sxl might act by associating with components of the general splicing machinery, the role of SNF has remained enigmatic. The role of SNF has been clarified by demonstrating that its contribution to the function of the U1 snRNP is not absolutely essential for viability of either sex, and that Sxl can associate with the U1 snRNP through a SNF-independent mechanism. Nevertheless, in vivo analysis continues to support a role for snf in Sxl splicing autoregulation by demonstrating that Sxl splicing defects are detectable under specific conditions. Interestingly, the phenotypic consequences of these Sxl splicing defects are more severe in the germline than in the soma. One possible explanation for this difference is that the requirements for Sxl splicing autoregulation are fundamentally different in the two tissue types. It is thought more likely that the mechanism is the same, but that the additional interaction with the U1 snRNP provided by SNF becomes critical when Sxl protein levels are low. This hypothesis is based on the fact that, in the germline, the majority of Sxl protein is cytoplasmic, and thus low levels of nuclear Sxl protein are the norm. By contrast, in other tissues, the Sxl protein accumulates in the nucleus, enabling the Sxl-U1 snRNP complex to form even when SNF is not stably associated with the U1 snRNP. The finding that these snf mutant females rarely survive if they are also heterozygous for Sxl, provides additional support for the idea that SNF function is only critical when Sxl protein levels are low (Nagengast, 2003).

Together, these studies argue that interactions between Sxl, the U1 snRNP and U2AF underlie the mechanism by which Sxl promotes skipping of the male exon. Based on these studies, a model is proposed in which Sxl acts not by preventing assembly of the U1 snRNP or U2AF onto the pre-mRNA, but instead interacts with the U1 snRNP bound to the male-exon 5' splice site, and U2AF at the male-exon 3' splice site, to form complexes that block these general splicing factors from assembling into a functional spliceosome. These 5' and 3' Sxl blocking complexes might function independently or they might interact across the exon to form a larger inhibitory complex. Furthermore, because it has not been possible to demonstrate that Sxl interacts directly with either U1-70K or U2AF, it is speculated that one or more bridging proteins are required to link Sxl to the general splicing machinery (Nagengast, 2003).

Although the in vivo approach cannot directly address when in the pathway of spliceosome assembly Sxl acts, biochemical studies have shown that during the course of spliceosome assembly, U2AF and the U1 snRNP are only transiently associated with splicing substrates, and are released before the formation of a functional spliceosome. Therefore, based on these studies, it seems reasonable to propose that Sxl acts by blocking splicing after splice site recognition but before catalysis begins. The data are therefore difficult to reconcile with the recent model, which proposes that Sxl blocks splicing after spliceosome assembly, at the second catalytic step of the reaction. Using RNA interference in Drosophila tissue culture cells it has been demonstrated that efficient male exon skipping depends on the presence of SPF45, a protein that is known to be required for the second step of splicing. Together with studies that show that SPF45 can bind to the upstream 3' splice site of the Sxl male exon and physically interact with Sxl, these data point to a role for SPF45 in Sxl splicing regulation. However, the primary evidence that Sxl blocks the splicing reaction during the second step rests on the results of in vitro splicing assays in which Sxl was shown to inhibit splicing of a chimeric splicing substrate that contains only a small region of the intronic region required for successful autoregulation in vivo. It is suspected that by looking at this 48 bp region, which contains a dispensable Sxl-binding site in addition to the two potential 3' splice sites, out of context, a failsafe mechanism was uncovered that comes into play when Sxl-mediated splicing regulation is otherwise compromised. Additional studies investigating the function of SPF45 in vivo will be required to determine the importance of this second step blocking mechanism and should provide insight into whether multiple mechanisms are needed to drive efficient regulated exon skipping (Nagengast, 2003).

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. This study attempts to identify factors that interact genetically and physically with fl(2)d. The Sxl-Fl(2)d protein-protein interaction was examined in detail and evidence is presented suggesting that the female-specific fl(2)d1 allele is antimorphic with respect to the process of sex determination. fl(2)d was shown to interact genetically with early acting general splicing regulators, and Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, no Fl(2)d complexes were detected containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, these findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly (Penn, 2008).

Alternative splicing of pre-mRNAs requires the default splicing machinery to choose between different potential 5' and 3' splice-site combinations. Factors like Sxl that force the selection of alternative 5' and 3' splice-site combinations must exert their effects through interactions with components of the general splicing machinery. However, since the splicing of pre-mRNAs is a multi-step process that depends upon the assembly and remodeling of a large and highly dynamic RNA protein complex, these regulatory interactions could potentially occur at many different points in the processing reaction. Previous genetic and molecular studies have implicated several general splicing factors in Sxl-dependent alternative splicing. These include Snf, U1-70k, U2AF, and Spf45. Of these proteins, only Snf is expected to be present in all of the intermediate steps in the splicing reaction. In contrast, studies on spliceosomal intermediates in humans indicate that the three other Sxl interactors are associated with the spliceosome only during the early stages of the splicing reaction (Jurica, 2003; Deckert, 2006). Both Snf and U1-70k are components of the U1 snRNP and will be present when the U1 snRNP first associates with the 5' splice site of the pre-mRNA to form the prespliceosome E complex. After U1 interacts with the 5' splice site, U2AF is thought to bind to the polypyrimidine tract upstream of the 3' splice site and recruit the U2 snRNP to the pre-mRNA to form spliceosome complex . In addition to Snf, U1-70k, and U2AF, this complex in humans also includes the SPF45 protein. In the next step, a complex containing three other snRNPs, the U4/U6,U5 tri-snRNP, associates with the spliceosome to form the B complex. This is followed by extensive structural rearrangements in which the U1 and subsequently U4 snRNPs are displaced. U170k together with the U1-associated Snf should be lost from the complex with disassociation of the U1 snRNP. The subsequent unwinding of the U4/U6 base pairs and dissociation of U4 permits base pairing between U6 and the 3' splice site and the U2 snRNA. This generates the active complex B*, which catalyzes the first transesterification reaction to generate complex C. Both U2AF and SPF45 appear to be missing from complex B*, while the only Sxl cofactor that is expected to remain until the final splicing step should be the Snf protein associated with the U2 snRNP. Thus, with the exception of Snf, the proteins known to be important for Sxl-dependent alternative splicing appear to function prior to the formation of the activated B* complex and the first splicing reaction (Penn, 2008).

Several lines of evidence suggest that this is also likely to be true for Fl(2)d. First, it was found that Fl(2)d is in an immunoprecipitable complex with Snf, U1-70K, and both of the U2AF subunits, U2AF50 and U2AF38. All of these proteins are expected to be present in one or more of the complexes (E, A, or B) that are formed early in the splicing reaction. Second, it has recently been found that U2AF not only is present in the early complexes E and A, but also can be detected in the inactive B complex (Deckert, 2006). Consistent with this expectation, complexes between U2AF50 and the U5 snRNP protein U5-40k were detected. In contrast, complexes between U2AF50 and Fl(2)d could be detected, complexes between could not be detected U5-40k and Fl(2)d. This finding would suggest that Fl(2)d is stably associated with the E and/or A complex, but is not stably associated with the B complex. Third, the SKIP protein associates with the inactive and activated B complexes, but is absent from the A complex (Jurica, 2003; Deckert, 2006). As was the case for U5-40k, interactions could not be detected between SKIP and Fl(2)d. With the caveat that these negative results must be interpreted with caution, these findings, taken together, would argue that Fl(2)d functions at an early step(s) in the splicing reaction prior to the formation of complex B (Penn, 2008).

Since Fl(2)d is expressed in both sexes and has functions in alternative splicing that are not connected to Sxl, it could be argued that the physical interactions detected between Fl(2)d and different components of the splicing apparatus do not reflect its functioning in Sxl-dependent alternative splicing. While this is a potential concern, there are a number of reasons why this is believed to be unlikely. For one, these complexes appear to be physiologically relevant to Sxl-dependent alternative splicing; female-specific genetic interactions were observed between fl(2)d and the genes encoding several of these proteins. In the case of snf, not only a null allele was tested, but also two mutations, snf148 and snf5mer, which differentially affect Snf protein interactions with the U1 or the U2 snRNPs, respectively. When mothers heterozygous for the U1-deficient snf148 are mated to Sxl- fathers, there is a marked reduction in the viability of female offspring. This female lethality is enhanced by the antimorphic allele fl(2)d1, but not by the null allele fl(2)d2. In contrast to snf148, there is little, if any, female-specific lethality in the progeny of snf5mer/+ females and Sxl- fathers; however, the snf5mer allele shows a very strong synergistic female lethal interaction with fl(2)d1. Likewise, a strong synergistic interaction was observed between fl(2)d1 and U2AF38δE18 (Penn, 2008).

Another reason to believe that the association of Fl(2)d with early splicing regulators is relevant to how it promotes Sxl-dependent alternative splicing is the fact that Sxl is found in complexes with the same set of splicing factors in nuclear extracts as Fl(2)d. As noted above, these factors include Snf, U170k, and the two U2AF subunits U2AF38 and U2AF50. In the case of Snf, it has been shown that Sxl is in an immunoprecipitable complex with Snf in nuclear extracts. Although this is also true for Fl(2)d, there are some differences in how Fl(2)d and Sxl interact with Snf. For one, Fl(2)d:Snf interactions in nuclear extracts are insensitive to RNase, while Sxl:Snf interactions are RNase sensitive. While it was not possible to test whether Fl(2)d:Snf interactions involve direct protein contacts, both Fl(2)d and Snf can interact directly with Sxl in vitro. Fl(2)d and Sxl also differ in their interactions with the Snf mutant proteins 148 and 5mer. Sxl can associate with the Snf5mer protein, but does not form a complex with Snf148. By contrast, Fl(2)d complex formation with the Snf148 mutant protein appears to be equivalent to that observed for wild-type Snf, while complexes with Snf5mer appear to be destabilized and are present in reduced yield. In addition, it was also found that Sxl resembles Fl(2)d in that it is not stably associated either with the U5 snRNP protein U5-40k or with SKIP (Penn, 2008).

On the basis of in vitro splicing experiments (using a chimeric pre-mRNA consisting of an adenovirus 5' exon/intron fused to a short sequence spanning the 3' splice site of the Sxl male exon) it has been suggestedthat Sxl autoregulation depends upon Sxl inhibition of the second catalytic step of splicing, i.e., the joining of the 5' splice site of the Sxl second exon to the 3' splice site of the male exon and the release of the second intron lariat intermediate. In this model, Sxl was proposed to block this catalytic step by inhibiting the SPF45 factor bound to one of the two AG sequences in the male exon 3' splice site. It was suggested that this would force the splicing machinery to bypass the male exon 3' splice site and instead join the free 5' splice site of exon 2 to the 3' splice of exon 4 located slightly more than a kilobase downstream of the male exon. In addition to the fact that using a 3' AG for the second catalytic step that is located ~1 kb from the branch point would be highly unusual, it is difficult to reconcile this model for Sxl autoregulation with the results presented in this study, which argue that Sxl must act at a much earlier point during the initial assembly of the splicing apparatus on target pre-mRNAs. Other findings also seem to be inconsistent with this model. For one, the Sxl-binding sites located in the polypyrimidine tract of the male exon 3' splice site that were used in the in vitro splicing experiments are completely dispensable for female splicing of the Sxl pre-mRNAs in vivo. In fact, the critical sites for Sxl binding are located in the upstream and downstream intron sequences flanking the male exon >200 bases from the male 3' splice site. In addition, Sxl regulation in vivo seems to pivot on blocking the use of the male exon 5' splice site, while controlling the use of the male exon 3' splice site plays at most only a subordinate role in regulation. Finally, although SPF45 is present in purified B spliceosome complexes from humans, it is apparently absent from the catalytically active B* and C complexes (Penn, 2008).

Another question of interest is the nature of the relationship between Sxl and Fl(2)d. Like Snf, Fl(2)d can interact directly with Sxl in vitro. For Snf, the first Sxl RRM domain R1 mediates this interaction while for Fl(2)d the interaction appears to depend upon a combination of the Sxl N terminus and the R1 RRM domain. Although the in vitro interactions between recombinant Sxl and Snf proteins are not dependent on (or stimulated by RNA), RNase treatment completely disrupts Sxl:Snf interactions in nuclear extracts. By contrast, RNase treatment appears to significantly enhance Sxl:Fl(2)d interactions in nuclear extracts. Since the two Sxl RRM domains undergo a substantial rearrangement when they bind to RNA, it is possible that Sxl:Fl(2)d interactions occur prior to the binding of the Sxl protein to the pre-mRNA, while Sxl:Snf interactions occur after Sxl has associated with its target sequences. If this is the case, then one plausible idea would be that Fl(2)d helps recruit Sxl into the assembling spliceosome. This mechanism could potentially account for the finding that fl(2)d mutations dominantly suppress the female lethal effects of the antimorphic Nβ-gal trangene: there would be less of Nβ-GAL fusion protein incorporated into the Sxl splicing complex when fl(2)d activity is reduced. However, since it was not possible to demonstrate an association between Fl(2)d and the Nβ-GAL fusion protein in vivo, other mechanisms for suppression cannot be excluded and further studies will be required to fully understand how Fl(2)d functions in Sxl-dependent alternative splicing (Penn, 2008).

Given that Sxl and key cofactors like Fl(2)d associate with early acting general splicing regulators that function to define the 5' and the 3' splice sites, it is possible that Sxl promotes female-specific splicing of its own pre-mRNAs by inhibiting the process of exon definition. Presumably it would do so by specifically targeting the U1 snRNP associated with the male exon 5' splice site and SPF45 and the U2AF heterodimer associated with the male exon 3' splice site. Exon definition is thought to be particularly important when a small exon is surrounded by large introns as is the case for the Sxl male exon. It is only ~190 bp in length and is flanked by large introns. Interestingly, exon definition cannot occur for exons >500 nucleotides and, if a large exon is surrounded by large introns, such an exon is often skipped entirely. Consistent with these studies, several of the gain-of-function mutations in Sxl are transposon insertions that increase the size of the male exon. In these mutants, Sxl is not required for female-specific splicing and the male exon is skipped, even in the absence of Sxl protein. Also supporting the notion that male exon definition might be an especially sensitive step that would make it a good target for Sxl regulation is the fact that both the 3' and 5' male exon splice sites are known to be suboptimal. In fact, when the male exon (plus the associated splice sites) is placed into a heterologous intron, the male exon is not recognized by the splicing machinery unless the splice sites are optimized to more closely resemble the consensus sequence. Even then, the male exon is not efficiently recognized by the default splicing machinery and is skipped most of the time. Further studies will be required to explore this possible mechanism for Sxl regulation (Penn, 2008).

Splicing mechanism of SXL: 4. Sxl autoregulation

The on/off state of the binary switch gene Sex-lethal is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the SXL proteins directs the splicing machinery to produce functional mRNAs. A blockage mechanism is employed in Sxl autoregulation. However, in contrast to Transformer, in which SXL appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in SXL splicing of its own mRNA. (i) Multiple cis-acting elements, both upstream and downstream of the male exon of SXL mRNA, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the SXL protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role (Horabin, 1993b).

SNF, a Drosophila homolog of mammalian U1A and U2B" snRNP proteins, is an integral component of the machinery required for splice site recognition in all pre-mRNAs. Mutations in snf disrupt the establishment of Sxl mRNA female-specific splicing pattern in both the germ line and soma. Because snf is required to establish the female-specific splice site choice, snf is likely to function in conjunction with SXL to bias the general splice machinery against the recognition of the male 5' splice site. SNF cooperates with SXL to block utilization of the male-specific exon of the SXL pre-mRNA, suggesting a model in which the SXL protein blocks spliceosome assembly by forming a non-productive snRNA/SXL complex. This suggests that snRNPs, like transcription factors, can have antagonistic roles in controlling gene expression (Flickinger, 1994 and Salz, 1996).

SXL and SNF proteins can interact directly in vitro, and these proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with anti-SXL antibody. The SNF protein associated with SXL protein is in a large, rapidly sedimenting complex. These complexes contain additional small nuclear ribonucleoprotein particle protein and the U1 and U2 small nuclear RNAs. Sxl transcripts can also be immunoprecipitated by anti-SXL antibodies. A model is presented for SXL mRNA splicing regulation. SXL protein binds to intron sequences far from the male exon (exon 3) and blocks the utilization of male exon splice sites. SXL proteins would bind to the poly U tracts in the introns upstream and downstream of the SXL male exon. SXL proteins in the intron would then make contact with the U1 and U2 snRNPs associated with the male exon splice junctions, presumably via protein-protein interactions with SNF. These contacts would prevent the snRNPs at the male exon splice junctions from participating in subsequent splicing steps (Deshpande, 1996).

Sex determination in Drosophila depends on the post-transcriptional regulatory activities of the gene Sex-lethal. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, a chimeric protein consisting of the N-terminal 99 amino acids fused to beta-galactosidase (hsp83:N beta-gal) was ectopically expressed. The Nbeta-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes (Deshpande, 1999a).

The fact that the phenotypic effects of the hsp83:N beta-gal transgene are sensitive to the relative dose of the endogenous Sxl gene argues that the beta-gal fusion protein acts like a classical dominant negative mutation, interfering with some critical regulatory activity of the Sxl protein. In principle, two different (but not mutually exclusive) mechanisms could account for the female lethality. The Nbeta-gal fusion protein could prevent the endogenous Sxl protein from properly downregulating the dosage compensation system. Alternatively, the fusion protein could turn off the endogenous Sxl gene by disrupting Sxl autoregulation. To determine if the Nbeta-gal fusion protein interferes with autoregulation, Sxl protein expression was compared in collections of wild-type and transgenic 0-12 hour embryos. The wild-type embryo population can be divided into two roughly equal classes based on the pattern of Sxl protein expression. Sxl protein is not expressed in male embryos, while female embryos express Sxl protein and are uniformly and darkly stained with Sxl antibody. A different result is obtained for the transgene embryos. (1) Instead of two classes, the transgene embryos can be divided into three classes: embryos that do not express Sxl protein and are, like wild-type males, unstained; embryos that express Sxl protein and, like wild-type females, are uniformly stained, and finally, embryos that express reduced levels of Sxl protein and show patchy antibody staining. (2) More than 60% of the transgenic embryos (instead of the expected 50%) fall in the unstained class while only about 30% of the embryos show the normal female staining pattern. These results indicate that Nbeta-gal fusion protein interferes with autoregulation. The expression of Msl-2 protein in wild-type and transgenic embryos. As expected, approximately 50% of the wild-type embryos express Msl-2 protein, while the other half do not. By contrast, 70% of the hsp83:N beta-gal transgene embryos are stained with the Msl-2 antibody, while only 30% are not. It is presumed that this latter group corresponds to transgenic female embryos that have wild-type Sxl protein expression. (Recall that 30% of the transgenic embryos have wild-type Sxl protein expression). Conversely, the former group should be composed of male embryos and of female embryos that either have no Sxl protein expression or have an abnormal pattern of Sxl protein expression. Taken together, these findings argue that the female lethal effects of the N beta-gal transgene arise, for the most part, indirectly through a disruption of Sxl autoregulation. Of course, the possibility that the fusion protein also antagonizes Sxl regulation of Msl-2 protein expression cannot be excluded (Deshpande, 1999a).

Since the Sxl gene can be completely deleted in males without any detectable effects on viability, morphology or behavior, the N beta-gal transgene would not, in principle, be expected to have any effect on male-specific developmental pathways. However, contrary to this expectation, approximately 10% of the males in all of the lines exhibit morphological alterations characteristic of sex transformations. These transformations include reduced or patchy abdominal pigmentation, extra sternite hairs, rotated genitalia and alterations in the number or shape of the sex combs. In females, the phenotypic effects of the hsp83:N beta-gal transgene can be enhanced by raising the temperature and suppressed by lowering the temperature. To test whether this is also true for male feminization, wild-type females were crossed to males heterozygous for the N-41 transgenic line, and examined the viability and morphology of the resulting transgenic animals at either 25°C or 18°C. The lethal effects of a single copy of the transgene on female progeny from this cross decreases from about 16% at the higher temperature to about 6% at the lower temperature. On the contrary, decreasing the temperature does not reduce the frequency of feminization; the number of partially feminized males increases from 7% to about 21% as the temperature is dropped from 25°C to 18°C. A low but readily detectable level of yp1 mRNA is present in transgenic N-172 and N-21 males, but not in the control wild-type males. Expression of yp1 mRNA in transgenic males does not require the Sxl gene but does require tra (Deshpande, 1999a).

The gain-of-function activity of the Nbeta-gal fusion protein is difficult to reconcile with the prevailing blockage model for the regulation of tra splicing. In this model, Sxl protein prevents the generic splicing factor U2AF from binding to the polypyrimidine tract of the default 3' splice site, forcing it instead to interact with the weaker downstream female-specific 3' splice site. A strong prediction of this model is that Sxl must be able to bind to the default polypyrimdine tract in order to block the binding of U2AF. Since specific binding to target RNAs requires the two Sxl RRM domains, the Nbeta-gal fusion protein should have absolutely no tra regulatory activity. This prediction is not fulfilled. A second, weaker, prediction is that the two Sxl RRM RNA-binding domains should be sufficient to regulate tra splicing. Taken together these findings call into question the simple blockage model and suggest that regulation of tra splicing may be different from that previously envisioned. The most likely mechanism is through interactions with generic RNA-binding proteins (or other components of the splicing machinery) that associate with TRA pre-mRNAs (Deshpande, 1999a).

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, SxlPe. The double dose of numerator elements in chromosomal females (XX) triggers transcription at SxlPe 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 effect interactions 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 U2'' 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/U2'' 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 SxlMf1 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).

Splicing regulation at the second catalytic step by Sex-lethal involves 3' splice site recognition by SPF45

Editorial note: A study by Nagengast (2003) and the study by Chaouki and Salz, 2006, describe below, suggest that the model proposed in the the Lallena study, described here, that concludes that Sxl blocks splicing after spliceosome assembly, at the second catalytic step of the reaction, is not correct.

The Drosophila protein Sex-lethal (Sxl) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with Sxl blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' splice site generated by a mutation that causes human beta-thalassemia (Lallena, 2002; full text of article).

Drosophila SPF45: A bifunctional protein with roles in both splicing and DNA repair

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, both of these studies have been unified and validated by demonstrating that the Drosophila protein is bifunctional, with independent functions in DNA repair and splicing. SPF45 associates with the U2 snRNP and mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with these finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair (Chaouki, 2006; full text of article).

Sex-lethal promotes female development by negatively regulating the N-signaling pathway

Notch signaling is used for cell-fate determination in many different developmental contexts. This study shows that the master control gene for sex determination in Drosophila, Sex-lethal, negatively regulates the N-signaling pathway in females. In genetic assays, reducing Sxl activity suppresses the phenotypic effects of N mutations, while increasing Sxl activity enhances the effects. Sxl appears to negatively regulate the pathway by reducing N protein accumulation, and higher levels of N are found in Sxl clones than in adjacent wild-type cells. The inhibition of N expression does not depend on the known downstream targets of Sxl; however, it was found that Sxl protein can bind to N mRNAs. Finally, these results indicate that downregulation of the N pathway by Sxl contributes to sex-specific differences in morphology and suggest that it may also play an important role in follicle cell specification during oogenesis (Penn, 2007).

While it has long been known that Sxl must control some aspects of sexual dimorphism by mechanisms that are independent of the Sxl→tra→dsx-fru regulatory cascade, understanding of what these morphological features might be and of how this might be accomplished has remained rudimentary. In these studies reported here, a regulatory link has been uncovered between Sxl and the N-signaling pathway. Sxl impacts the functioning of this pathway in a sex-specific fashion by negatively regulating N itself (Penn, 2007).

Several lines of evidence support the conclusion that the N-signaling pathway is a target for Sxl regulation. N and Sxl show genetic interactions in a variety of different developmental contexts. In the ovary, egg-chamber packaging defects are induced when homozygous Nts1 females are placed at the nonpermissive temperature. Eliminating one copy of the Sxl gene dominantly suppresses these egg-chamber packaging defects. In female wing discs, N is haploinsufficient for the formation of the tip of the wing blade. This haploinsufficiency is sensitive to the Sxl gene dose. The N wing phenotype is suppressed when females have only one functional Sxl gene, while it is exacerbated when females have three functional Sxl genes. Like wing development, N is 'haploinsufficient' in females for bristle formation in the A5 sternite, and bristle number is increased in heterozygous flies. This bristle phenotype is suppressed when the N/+ females have only one Sxl gene, while it is enhanced when the females have three Sxl genes. Finally, the female lethal effects of a combination of loss of function N alleles can be suppressed by reducing the Sxl dose. Taken together, these genetic interactions argue that Sxl must negatively regulate the N pathway. Moreover, in each of these contexts, the regulatory interactions between Sxl and N must be independent of both the Sxl→tra→dsx-fru regulatory cascade and of the msl dosage compensation system. The reason for this is that Sxl is not haploinsufficient for either tra splicing or for turning off the msl-2 dosage compensation system, and in females heterozygous for Sxl, both of these regulatory pathways are fully in the female mode. Likewise, adding an extra dose of Sxl would not hyperfeminize tra nor would it further repress msl-2 translation. In this context, it should also be pointed out that Sxl negatively regulates its own expression through binding sites in the UTRs of Sxl mRNAs. Because of this negative autoregulatory feedback loop, the levels of Sxl protein in both Sxl/+ and Sxldup/+ females are maintained close to that in wild-type females. Thus, the effects of Sxl on N activity are likely to be underestimated in genetic interaction experiments (Penn, 2007).

These is a substantial upregulation of N protein in Sxl follicle clones. This upregulation is independent of the Sxl→tra→dsx-fru regulatory cascade; however, in this case, it is suspected that two factors likely contribute to the observed increase in N protein. The first is the loss of Sxl regulation, while the second is the activation of the msl-2 dosage compensation system in the complete absence of Sxl activity. As the latter is expected to generate only a 2-fold increase in N expression, it would not fully account for the effects of losing Sxl activity in the clones (c.f., the N levels in adjacent stage 10 Sxl+ and Sxl follicle cells) (Penn, 2007).

Finally, like the two known targets for translational regulation by Sxl, msl-2, and Sxl, N mRNA has multiple Sxl binding sites in its UTRs. Moreover, as would be expected if Sxl directly downregulated N protein accumulation by controlling the translation of N message, Sxl binds to N mRNAs in ovaries. It is interesting to note that the configuration of Sxl binding sites in N mRNAs is quite similar to msl-2. Both mRNAs have two Sxl binding sites in the 5′UTR and four in the 3′UTR. In spite of the similarity in the number and distribution of Sxl binding sites, Sxl repression of N mRNA translation must differ from its repression of msl-2 mRNA translation because unlike Msl-2, N protein is expressed in females. One factor that might account for this difference is that repression of msl-2 mRNA translation by Sxl depends upon corepressors that interact with sites in the 3′UTR located adjacent to the Sxl binding sites; however, these putative corepressor recognition sequences are not present next to the Sxl binding sites in the N 3′UTR (Penn, 2007).

The N signaling pathway plays a central role in fly development because of its ability to specify alternative cell fates. Since most of the tissues and cell types in which the N pathway functions are present in both males and females, an obvious question is how Sxl can deploy this pathway to generate sex-specific differences in morphology. The results indicate that in common tissues, Sxl is able to generate sex-specific differences by changing the level of N activity. Thus, in the A5 sternite, the number of bristles in females is greater than in males, and this difference is due to the downregulation of N by Sxl in female flies. As in other parts of the adult cuticle, bristle formation in A5 depends upon the level of N activity. The number of bristles is inversely proportional to N activity, and N heterozygous females have a greater number of bristles than wild-type females. This difference can be suppressed by reducing Sxl activity and magnified by increasing Sxl activity. Excess Sxl activity can also cause an increase in the number of A5 bristles in females that are wild-type for N. It is reasonable to suppose that this general downregulation of N by Sxl will contribute to other morphological differences between males and females that are independent of the Sxl→tra→dsx-fru regulatory cascade such as bristle number in other parts of the adult cuticle, size of tissues and organs, and perhaps some as yet unknown aspects of nervous system development (Penn, 2007).

Since the ovary is only present in females the developmental context for Sxl-N regulatory interactions is different from most other tissues in the fly. Like the wing and sternites, Sxl negatively regulates N in the ovarian follicular epithelium. When Sxl activity is lost in follicle cells, a variety of defects were observed in the development of this epithelium, including egg-chamber packaging defects, ectopic polar cells, and extra-long interfollicular stalks. This spectrum of phenotypes closely resembles those seen when there is excess N activity and argues that N must be inappropriately upregulated in the follicular epithelium when Sxl is lost. Consistent with this suggestion, elevated levels of N protein are found in Sxl clones. With the possible caveat that the MSL dosage compensation system is likely activated in the absence of Sxl and thus probably contributes to the upregulation of N protein, these observations suggest that Sxl plays an important role in mediating N specification of cell fate as the follicular epithelium develops. This view is supported by the reciprocal patterns of N and Sxl protein accumulation in the germarium of wild-type females. Follicle cells expressing high levels of N in the germarium have only little cytoplasmic Sxl, while lower levels of N are found in follicle cells that have high amounts of cytoplasmic Sxl. If, as is suspected, Sxl regulates N at the level of translation, the turnover of cytoplasmic Sxl and/or its relocalization to the nucleus would be expected to lead to the upregulation of N protein expression. Conversely, in cells that retain abundant cytoplasmic Sxl, N expression should remain repressed. Since the cells in the germarium that are induced to express high levels of N are thought to be the progenitors of the stalk and polar cells, releasing N mRNA from translation inhibition by Sxl would be expected to facilitate the specification of these cell types by the N-signaling pathway (Penn, 2007).

This raises the question of why cytoplasmic Sxl turns over and/or is targeted to the nucleus in these particular cells. In the germline and in the wing disc, turnover and nuclear targeting of Sxl protein are known to be mediated by the hh signaling pathway. It seems possible that hh signaling might also promote the turnover/nuclear targeting of Sxl in these particular somatic follicle cells. Consistent with this idea, overexpression of hh in follicle cells leads to at least one of the phenotypes that is seen when Sxl activity is lost (or N is ectopically activated), the expansion of interfollicular stalks. If hh is responsible for the turnover/nuclear targeting of Sxl, the Sxl gene would provide a mechanism for linking the hh- and N-signaling pathways in the specification of stalk and polar cell fates. Further studies will be required to test this model (Penn, 2007).

Structural basis for the assembly of the Sxl-Unr translation regulatory complex

Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. This study reports the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl-Unr-msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. These results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression (Henning, 2014).

Translational repression of msl2 mRNA is coordinated by Sxl binding to uridine-rich stretches in both untranslated regions (UTRs): binding to the 3' UTR inhibits the recruitment of the small ribosomal subunit whereas binding to the 5' UTR inhibits the scanning of those subunits that presumably have escaped the 3' UTR-mediated control. At the 3' UTR, the recruitment of Unr by Sxl to bind in close spatial proximity is critical for translational repression. The region of Sxl containing residues 122-301 (Drosophila RNA binding domain 4, dRBD4) shows full translational repression activity, while the RNA recognition motifs (RRMs) alone (residues 123-294, dRBD3) are necessary and sufficient for RNA binding. Only the first cold-shock domain (CSD1) of Unr is required for complex formation with Sxl and msl2 mRNA. Notably, CSD1 and Sxl do not interact in the absence of RNA, suggesting a cooperative binding mechanism. A 46-nucleotide region in the msl2 3' UTR containing two uridine-rich Sxl-binding sites is sufficient for complex formation and translational repression. To identify the minimal region required for Unr and Sxl binding, ternary complex formation was analysed by electrophoretic mobility shift assays (EMSA) using wild-type and variant RNAs. Binding of dRBD4 and Unr to the wild-type RNA indicated the presence of two complexes. The number of complexes was reduced to one by mutation of either Sxl-binding site, and site F supported complex formation with a higher affinity than site E. Mutation of the sequences surrounding site F affected Unr binding, while more distal mutations did not impair complex formation. These data indicate the formation of 2:2:1 and 1:1:1 dRBD4-Unr-RNA complexes representing the two bands of slower mobility, which was further confirmed by static light scattering measurements (Henning, 2014).

Taken together these data demonstrate that the triple zipper and the non-canonical RNA contacts mediated by Sxl RRM1 are critical for translational regulation by Sxl and Unr. It is important to note that these interactions are essential for msl2 translational repression, but are dispensable for the regulation of transformer pre-mRNA splicing19, as recognition of the uridine-rich 5' region of msl2 RNA by Sxl dRBD3 in the ternary complex is virtually identical to that previously observed for transformer pre-mRNA11. Therefore, recognition of a uridine-rich RNA sequence by Sxl can play distinct roles in regulating splicing and translation depending on the binding of Unr in close proximity (Henning, 2014).

The data also explain why human Unr can form a complex with Drosophila Sxl and msl2 RNA as all residues involved are conserved (His 213, Asp 237 and Arg 239). In contrast, CSD1 alone can bind a variety of distinct RNA sequences with similar affinity in the absence of Sxl. This indicates that strong and specific RNA recognition for the GCACG motif in msl2 RNA depends on the presence of Sxl in the ternary complex. Interestingly, C11 does not conform to the previously reported consensus sequence for human Unr CSD1, but is nevertheless strictly conserved in the msl2 mRNA of organisms that may employ D. melanogaster-like dosage compensatio. Consistent with this, CSD1 Asp 237 and Arg 239, which recognize C11, are conserved in CSD1 but not in CSD2-5 of Unr proteins or in cold shock domains of other proteins (Henning, 2014).

Although the Drosophila dosage-compensation mechanism is not conserved in mammals, it is expected that ternary interactions involving RRM and CSD domains with RNA may be important for other biological functions. For example, human orthologues of the proteins examined in this study, such as the Sxl orthologue HuR or the RNA binding protein RBM6, share triple-zipper and α1-helix residues, which could mediate similar interactions (Henning, 2014).

Sandwiching of single-stranded RNA by multiple proteins has been observed previously, for example in small nuclear ribonucleic particles or the exon junction complex, but the intertwined recognition observed in this study is particularly intriguing. Moreover, the combination of these two general and abundant RNA binding domains (RRM and CSD), which are also involved in other RNA binding events, generates a new and unique binding specificity for single-stranded RNA. The intertwined cooperative binding of Sxl and Unr establishes a functionally active arrangement of multiple RNA binding domains from two distinct proteins, thus extending principles recently observed for multi-domain RNA binding proteins (Henning, 2014).

These results show that repression of a biological process with dramatic consequences for viability depends on the establishment of a specific set of novel molecular interactions. This is of particular significance considering that a limited set of RNA binding modules has been identified in the mRNA interactome. The Unr-Sxl-msl2 complex illustrates how the combinatorial activity of general RNA binding domains expands the code for RNA recognition by establishing unique and distinct ribonucleoprotein architectures and thus greatly amplifying the opportunities for regulation of gene expression (Henning, 2014).

Table of contents

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

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