Sex lethal
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).
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).
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).
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).
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).
The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes (Smolko, 2018).
In metazoans, germ cell development begins early in embryogenesis when the primordial germ cells are specified as distinct from somatic cells. Specified primordial germ cells then migrate into the embryonic gonad, where they begin to exhibit sex-specific division rates and gene expression programs, ultimately leading to meiosis and differentiation into either eggs or sperm. Defects in sex-specific programming interferes with germ cell differentiation leading to infertility and germ cell tumors. Successful reproduction, therefore, depends on the capacity of germ cells to maintain their sexual identity in the form of sex-specific regulation of gene expression (Smolko, 2018).
In Drosophila melanogaster, germ cell sexual identity is specified in embryogenesis by the sex of the developing somatic gonad. However, extrinsic control is lost after embryogenesis and sexual identity is preserved by a cell-intrinsic mechanism. The Sex-lethal (Sxl) female-specific RNA binding protein is an integral component of the cell-intrinsic mechanism, as loss of Sxl specifically in germ cells leads to a global upregulation of spermatogenesis genes and a germ cell tumor phenotype. Remarkably, sex-inappropriate transcription of a single gene, PHD finger protein 7 (phf7), a key regulator of male identity, is largely responsible for the tumor phenotype. Depletion of phf7 in mutants lacking germline Sxl suppresses the tumor phenotype and restores oogenesis. Moreover, forcing PHF7 protein expression in ovarian germ cells is sufficient to disrupt female fate and give rise to a germ cell tumor. Interestingly, sex-specific regulation of phf7 is achieved by a mechanism that relies primarily on alternative promoter choice and transcription start site (TSS) selection. Sex-specific transcription produces mRNA isoforms with different 5' untranslated regions that affect translation efficiency, such that PHF7 protein is only detectable in the male germline. Although the Sxl protein is known to control expression post-transcriptionally in other contexts the observation that germ cells lacking Sxl protein show defects in phf7 transcription argues that Sxl is likely to indirectly control phf7 promoter choice. Thus, how this sex-specific gene expression program is stably maintained remains to be determined (Smolko, 2018).
This study reports the discovery that female germ cell fate is maintained by an epigenetic regulatory pathway in which SETDB1 (aka EGGLESS, KMT1E, and ESET) is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. SETDB1 trimethylates H3K9 (H3K9me3), a feature of heterochromatin. Using tissue-specific knockdown approaches this study established that germ cell specific loss of SETDB1, its protein partner WINDEI [WDE, aka ATF7IP, MCAF1 and hAM10], and the H3K9me3 reader, HP1a, encoded by the Su(var)205 locus, leads to ectopic expression of euchromatic protein-encoding genes, many of which are normally expressed only in testis. It was further found that H3K9me3 repressive marks accumulate in a SETDB1 dependent manner at 21 of these ectopically expressed genes, including phf7. Remarkably, SETDB1 dependent H3K9me3 deposition is highly localized and does not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, where H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific TSS. Lastly, this study found that H3K9me3 accumulation at many of these genes, including phf7, is dependent on Sxl. Collectively these findings support a model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes (Smolko, 2018).
This study reveals a role for H3K9me3 chromatin, operationally defined as facultative heterochromatin, in securing female identity by silencing lineage-inappropriate transcription. H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, are required for silencing testis gene transcription in female germ cells. These studies further suggest a mechanism in which SETDB1, in conjunction with the female fate determinant Sxl, controls transcription through deposition of highly localized H3K9me3 islands on a select subset of these genes. The male germ cell sexual identity gene phf7 is one of the key downstream SETDB1 target genes. H3K9me3 deposition on the region surrounding the testis-specific TSS guaranties that no PHF7 protein is produced in female germ cells. In this model, failure to establish silencing leads to ectopic PHF7 protein expression, which in turn drives aberrant expression of testis genes and a tumor phenotype (Smolko, 2018).
Prior studies have established a role for SETDB1 in germline Piwi-interacting small RNA (piRNA) biogenesis and TE silencing. However, piRNAs are unlikely to contribute to sexual identity maintenance as mutations that specifically interfere with piRNA production, such as rhino, do not cause defects in germ cell differentiation. These findings, together with the observation that rhino does not control sex-specific phf7 transcription, suggests that the means by which SETDB1 methylates chromatin at testis genes is likely to be mechanistically different from what has been described for piRNA-guided H3K9me3 deposition on TEs.
The accumulation of H3K9me3 at many of these genes, including phf7, is dependent on the presence of Sxl protein. Thus, these studies suggest that Sxl is required for female-specific SETDB1 function. Sxl encodes an RNA binding protein known to regulate its target genes at the posttranscriptional levels. Sxl control may therefore be indirect. However, studies in mammalian cells suggest that proteins with RNA binding motifs are important for H3K9me3 repression, raising the tantalizing possibility that Sxl might play a more direct role in governing testis gene silencing. Further studies will be necessary to clarify how the sex determination pathway feeds into the heterochromatin pathway (Smolko, 2018).
phf7 stands out among the cohort of genes regulated by facultative heterochromatin because of its pivotal role in controlling germ cell sexual identity. Because ectopic protein expression is sufficient to disrupt female fate, tight control of phf7 expression is essential. phf7 regulation is complex, employing a mechanism that includes alternative promoter usage and TSS selection. This study reports that SETDB1/H3K9me3 plays a critical role in controlling phf7 transcription. In female germ cells, H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific transcription start site. Dissolution of the H3K9me3 marks via loss of Sxl or SETDB1 protein is correlated with transcription from the upstream testis-specific site and ectopic protein expression, demonstrating the functional importance of this histone modification. Together, these studies suggest that maintaining the testis phf7 promoter region in an inaccessible state is integral to securing female germ cell fate (Smolko, 2018).
Although the loss of H3K9me3 pathway members in female germ cells leads to the ectopic, lineage-inappropriate transcription of hundreds of genes, integrative analysis identified only 21 SETDB1/H3K9me3 regulated genes. Given that one of these genes is phf7 and that ectopic PHF7 is sufficient to destabilize female fate, it is likely that inappropriate activation of a substantial number of testis genes is a direct consequence of ectopic PHF7 protein expression. How PHF7 is able to promote testis gene transcription is not yet clear. PHF7 is a PHD-finger protein that preferentially binds to H3K4me2, a mark associated with poised, but inactive genes and linked to epigenetic memory. Thus, one simple model is that ectopic PHF7 binds to H3K4me2 marked testis genes to tag them for transcriptional activation (Smolko, 2018).
It will be interesting to explore whether any of the other 20 SETDB1/H3K9me3 regulated genes also have reprogramming activity. In fact, ectopic fate-changing activity has already been described for the homeobox transcription factor Lim1 in the eye-antenna imaginal disc. However, whether Lim1 has a similar function in germ cells is not yet known. Intriguingly, protein prediction programs identify three of the uncharacterized testis-specific genes as E3 ligases. SkpE is a member of the SKP1 gene family, which are components of the Skp1-Cullin-F-box type ubiquitin ligase. CG12477 is a RING finger domain protein, most of which are believed to have ubiquitin E3 ligases activity. CG42299 is closely related to the human small ubiquitin-like modifier (SUMO) E3 ligase NSMCE2. Given studies that have linked E3 ligases to the regulation of chromatin remodeling, it is tempting to speculate that ectopic expression of one or more of these E3 ligases will be sufficient to alter cell fate. Future studies focused on this diverse group of SETDB1/H3K9me3 regulated genes and their role in reprogramming may reveal the multiple layers of regulation required to secure cell fate (Smolko, 2018).
The SETDB1-mediated mechanism for maintaining sexual identity uncovered in this study may not be restricted to germ cells. Recent studies have established that the preservation of sexual identity is essential in the adult somatic gut and gonadal cells for tissue homeostasis. Furthermore, the finding that loss of HP1a in adult neurons leads to masculinization of the neural circuitry and male specific behaviors suggests a connection between female identity maintenance and H3K9me3 chromatin. Thus, it is speculated that SETDB1 is more broadly involved in maintaining female identity (Smolko, 2018).
These studies highlight an emerging role for H3K9me3 chromatin in cell fate maintenance. In the fission yeast S. pombe, discrete facultative heterochromatin islands assemble at meiotic genes that are maintained in a silent state during vegetative growth. Although less well understood, examples in mammalian cells indicate a role for SETDB1 in lineage-specific gene silencing. Thus, silencing by SETDB1 controlled H3K9 methylation may be a widespread strategy for securing cell fate. Interestingly, H3K9me3 chromatin impedes the reprogramming of somatic cells into pluripotent stem cells (iPSCs). Conversion efficiency is improved by depletion of SETDB1. If erasure of H3K9me3 via depletion of SETDB1 alters the sexually dimorphic gene expression profile in reprogrammed cells, as it does in Drosophila germ cells, the resulting gene expression differences may cause stem cell dysfunction, limiting their therapeutic utility (Smolko, 2018).
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).
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Female fate maintenance in Drosophila germ cells: The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells
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