Sex lethal


REGULATION


Table of contents

Determination of male somatic sex and function of Sxl in XY germ cells

Dosage compensation in Drosophila requires the male-specific lethal (msl) proteins (Msl) to make gene expression from the single male X chromosome equivalent to that from both female X chromosomes. Expression of ms12 is repressed post-transcriptionally by Sex lethal (Sxl). Although MSL2 mRNA is alternatively spliced in males and females, this does not alter its coding potential and splicing is not required for male-specific expression of Msl2 protein. Instead, these results suggest that the association of Sxl protein with multiple sites in the 5' and 3' untranslated regions of the MSL2 transcript represses its translation in females. Thus, this well characterized alternative splicing factor regulates at least one target transcript by a distinct mechanism (Kelley, 1997).

Sex-lethal acts synergistically through sequences in both the 5' and 3' untranslated regions of MSL-2 to mediate repression. There is a small intron in the 5' UTR of MSL-2 pre-messenger RNA that is retained in females and removed in males (the male specific intron). Additionally, there are poly (U) runs in both the 5' and 3' UTRs that resemble the SXL-binding sites in SXL and TRA. The male-specific intron present in the 5' UTR of female mRNA functions primarily to allow SXL binding. Removal of either the 3' or both the 3' and 5' UTRs result in expression of MSL-2 protein in females. Endogenous MSL-2 mRNA is not retained in the nucleus of wild-type animals. Two possible mechanisms of SXL-mediated posttranscriptional regulation have been proposed: (1) that SXL binds MSL-2 mRNA and prevents its export from the nucleus, and (2) that SXL binds MSL-2 mRNA and directly blocks its translation. The observation that MSL-2 mRNA is predominantly localized in the cytoplasm of both males and females argues in favor of a direct role for SXL in translational regulation. This direct role has been verified by establishing that the levels of mRNA in the cytoplasm are not influences by the presence or absence of SXL protein. This is the first characterized instance of an mRNA that has target sequences for the same translational regulatory factor in both the 5' and 3' UTRs (Bashaw, 1997).

In females, SXL functions to negatively regulate MSL-2 at the levels of mRNA processing and translational control such that no MSL-2 is produced in females. MSL-2 protein is co-localized with the other three MSL proteins at hundreds of sites along the male polytene X chromosome. In the absense of MSL-2 the other three male specific chromosomal silencing proteins, MSL-1, MSL-3 and MLE cannot bind to chromosomes. msl-2 encodes a protein with a putative DNA-binding domain: the RING finger. MSL-2 protein is not produced in females and sequences in both the 5' and 3' UTRs are important for this sex-specific regulation. Furthermore, MSL-2 pre-mRNA is alternatively spliced in a Sex-lethal-dependent fashion in its 5' UTR. (Bashaw, 1995).

MSL-2 consists of 769 amino acid residues and has a RING finger (C3HC4 zinc finger) and a metallothionein-like domain with eight conserved and two non-conserved cysteines. In addition, it contains a positively and a negatively charged amino acid residue cluster and a coiled coil domain that may be involved in protein-protein interactions. Males produce a MSL-2 transcript that is shorter than in females, due to differential splicing of an intron of 132 bases in the untranslated leader. The protein is expressed at a detectable level only in males, where it is physically associated with the X chromosome. MSL-2 may be the target of the master regulatory gene Sxl (Zhou, 1995).

In Drosophila, the single male X chromosome is transcribed at twice the rate of a single female X chromosome. This hypertranscription requires the functions of at least four autosomal male-specific lethal genes (msls) and is under the control of the Sex-lethal gene. One of the msls, the maleless (mle) gene, encodes a protein that is associated with the male X chromosome. In females, SXL functions indirectly to prevent MLE from binding to the two X chromosomes. Additionally, MLE X chromosome binding requires wild-type msl1, msl2, and msl3 functions. These data support a model whereby the activity of the MLE protein is regulated through its association with one or more of the other MSL proteins (Gorman, 1993).

Dosage compensation in Drosophila occurs by a twofold increase in transcription per copy of X-linked genes in males (XY) compared with females (XX). MSL-1 is one of four genes that are essential for dosage compensation in males, and MSL-1 protein is associated specifically with the male X chromosome. MSL-1 protein levels are negatively regulated by SXL in females, resulting in male-specific expression of MSL-1. In addition, msl-2 is required for translation and/or stability of MSL-1 in males. Furthermore, the wild-type pattern of MSL-1 localization to the X chromosome is dependent on mle and msl-3 function, although a subset of sites are stained with MSL-1 antibodies in these mutants (Palmer, 1994).

At least some aspect of dosage compensation is not carried out by Male-specific-lethals, including MSL-2. Early runt dosage compensation is directed by the product of the early promoter of Sex lethal. Thus the early transcripts of Sex lethal have a role in addition to splicing, that is, in directing the early stages of dosage compensation. runt dosage compensation is a consequence of early Sex lethal expression in females. Since MSL-1 and MSL-2 begin to associate with the X chromosome during the cellular blastoderm, it is likely that MSL-independent compensation of genes such as runt and MSL-mediated compensation of other early-acting X-linked genes could either be separated by a very short developmental period or could occur simultaneously. The mechanism of Sex lethal directed early dosage compensation is unknown (McDowell, 1996 and Bernstein, 1994).

Presence of functional Sex lethal is responsible for the absence of RNA on the X-1 RNA in females. As presence of functional Sex lethal protein in females is responsible for a blocking of functional MSL-2 splicing in females, mutation of Sxl results in functional MSL-2 splicing in females and production of roX1 levels comparable to those observed in wild-type males (Meller, 1997).

Male-specific expression of the protein Male-specific-lethal 2 (Msl-2) controls dosage compensation in Drosophila. Msl-2 gene expression is inhibited in females by Sex-lethal (Sxl), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of MSL-2 mRNA contains putative Sxl binding sites and is retained in female flies. Sxl plays a dual role in the inhibition of MSL-2 expression. Cotransfection of Drosophila Schneider cells with a Sxl expression vector and a reporter containing the 5' UTR of MSL-2 mRNA results in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation is blocked by Sxl, but not by the intron per se. Both splicing and translation inhibition by Sxl were recapitulated in vitro and found to be dependent on Sxl binding to high-affinity sites within the intron, showing that Sxl directly regulates these events. These data reveal a coordinated mechanism for the regulation of MSL-2 expression by the same regulatory factor: Sxl enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm (Gebauer, 1998).

Translational repression of male-specific-lethal 2 (MSL-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between Sxl-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of MSL-2 translational control, a novel cell-free translation system has been developed from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of MSL-2 translation in this system requires cooperation between the Sxl-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of MSL-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by Sxl (Gebauer, 1999).

Translational regulation of MSL-2 mRNA in vitro primarily rests on the cooperation between Sxl-binding site B in the 5' UTR and the sites located in the 3' UTR. The presence of functional sites in only one of the UTRs results in a 2-fold inhibition by Sxl, while the presence of sites in both UTRs achieves greater than 10-fold inhibition. This suggests that Sxl action from the 5' and 3' UTR sites cooperates to prevent translation. One possible scenario is that Sxl-driven interactions between the 5' and 3' UTRs package the MSL-2 mRNP into a conformation that renders it poorly accessible to the translational machinery. An alternative explanation is that Sxl inhibits different processes at the 5' and 3' UTRs independently, but that these processes cooperate for translation. This model would not pose any a priori requirement for interactions between Sxl molecules bound to the two ends of MSL-2 mRNA. A major result of this work is the realization that Sxl action is independent of the presence of the poly(A) tail, at least in vitro. The cordycepin-treated mRNA contains a small fraction of messages with a poly(A) tail of 30 residues. These do not appear to contribute significantly either to overall translation or to regulation by Sxl, because the non-treated mRNA, which contains a higher proportion of A30 molecules, is translated and repressed by Sxl with comparable efficiency. Importantly, the Drosophila embryo system is principally poly(A) dependent. Since it so closely reflects the known aspects of MSL-2 regulation in vivo, it is predicted that poly(A) independence is an important feature of how Sxl regulates dosage compensation in the fly. The Drosophila cell-free translation system described here now allows the study of an important aspect of developmental biology in the test tube. Future experiments using this system should help to shed further light on the mechanism by which Sxl inhibits translation. Equally importantly, it provides a novel approach to dissect other examples of translational regulation during Drosophila embryogenesis, and to study the mechanism by which the poly(A) tail promotes translation in multicellular eukaryotes (Gebauer, 1999).

When XX germ cells develop in a testis they become spermatogenic. Thus, somatic signals determine the sex of genetically female germ cells. In contrast, XY germ cells experimentally transferred to an ovary do not differentiate oogenic cells. Because such cells show some male characteristics when analyzed in adults, it was assumed that XY germ cells autonomously become spermatogenic. However, recent evidence has shown that a female soma feminizes XY germ cells (Waterbury, 2000). The conclusion was drawn that the sex determination of XY germ cells is dictated by the sex of the soma. The fate of XY germ cells placed in a female environment has been monitored throughout development. Such germ cells respond to both cell-autonomous and somatic sex-determining signals, depending on the developmental stage. Analyzing the expression of sex-specific molecular markers, autonomous male-specific gene expression was first detected in XY germ cells embedded in female embryos and larvae. At later stages, however, sex-specific regulation of gene expression within XY germ cells is influenced by somatic gonadal cells. After metamorphosis, XY germ cells developing in a female soma start expressing female-specific and male-specific markers. Transcription of female-specific genes is maintained, while that of male-specific genes is later repressed. In such XY germ cells, the female-specific gene Sex-lethal is activated. Within the germline, Sxl expression is required for the activation of a further female-specific gene and the repression of male-specific genes. The existence of downstream targets of the gene Sxl in the germline is reported here, for the first time (Janzer, 2001).

A female soma feminizes XY germ cells during larval and pupal phases. During the feminization process, male-specific genes are repressed and Sxl is activated. Sxl in turn activates the female marker Q13d. The feminization occurs in spite of the presence of male gene products, such as male fusome structures and male-specific crystals, which are formed during larval stages when XY germ cells autonomously express male-specific genes. Therefore, male and female products are found in such cells. The occurrence of both male and female gene expression was initially not understood. Many scientists have tried to analyze the sex of abnormal germ cells, for example germ cells carrying mutations of Sxl, by amplifying sex-specific gene products by RT-PCR. In these experiments, ovaries containing abnormal germ cells are pooled and the presence of transcripts is monitored. The results presented here show that any sex-specific probe is expected to be amplified in such an experiment. This, however, will not reveal the sexual identity of the individual tested cells, and in particular it will not show changes in sex-specific gene expression as flies age. In this study, sex-specific gene expression was tested for individually, in single cells, and animals were analyzed at different stages of development. Therefore, it was possible to observe that individual cells can express male- or female-specific genes and that the ability of cells to respond to sex-determining somatic signals depends on the X:A ratio and the developmental stage (Janzer, 2001).

In undifferentiated germ cells of XX ovaries, Sxl protein is present as a ubiquitous cytoplasmic molecule. In later stages, the protein is also found in nuclei localized in specific foci. So far, no specific function has been assigned to the cytoplasmic nor to the nuclear Sxl protein. Furthermore, no target of Sxl is known in the germline. Targets of Sxl, such as tra or msl-2, or dsx which is indirectly controlled by Sxl, have been identified, but these targets are all controlled by somatic Sxl. It has been shown here that Q13d is a target of Sxl, and that cytoplasmic but not the localized nuclear Sxl protein is required for the activation of Q13d. It is predicted that the activation by Sxl is indirect, because female-specific transcription is monitored. Sxl, an RNA-binding protein, does not regulate the transcription of genes. Rather, acting post-transcriptionally in somatic cells, it controls the alternative splicing of the sex-determining gene tra and represses translation of Msl-2, which is required to obtain a male-specific level of expression of X-linked genes. Furthermore, Sxl has been postulated to interact with specific sites of early expressed X-chromosomal RNAs to achieve an early msl-2 unrelated form of dosage compensation (Janzer, 2001 and references therein).

The germline function of Sxl is required for the activation of a female and the repression of several male markers. The results presented here show that male gene activity is gradually repressed during larval stages in germ cells of XY pseudofemales. Nevertheless, transient expression of male markers is found in adults. After hatching, germ cells of XY pseudofemales strongly proliferate to yield multicellular cysts. It is likely that sex-determining somatic signals are formed, but that these signals cannot reach all the rapidly proliferating cells immediately. This results in a transient loss of sex-specific gene regulation that is only reestablished later, when signals reach their target genes. A new function for the gene Sxl is reported here; within the germ-line Sxl is required to establish a somatically controlled sex-specific gene regulation (Janzer, 2001).

The maintenance of male-specific gene expression in the germline depends on interactions with a male soma. There are two alternative explanations as to why the expression of male-specific germline genes is progressively lost in XY germ cells that develop in ovaries. Somatic testis cells might send signals required for maintenance, or somatic ovarian cells might send signals to repress male-specific gene expression. In male and female embryos that do not have gonads, germ cells can express the male-specific enhancer-trap mgm1. This suggests that an ovary-derived repressing signal prevents male-specific gene expression in germ cells of wild-type females. However, not all germ cells express the male marker in XX embryos lacking gonads, and mgm1 expression is always weaker than in germ cells that are integrated into embryonic testes. Thus, a male somatic signal must enhance male-specific gene expression in germ cells. This shows that interactions between somatic gonadal cells and germ cells are of fundamental importance not only in XX but also in XY animals (Janzer, 2001).

tra-dependent signals, that must originate from somatic cells, activate female-specific and repress male-specific germline genes. This does not only happen in XY germ cells developing in an ovary, but also in XX germ cells. In XX females, the signals act earlier than in XY pseudofemales, since in wild-type ovaries Q13d expression is seen already in second instar larvae. This is probably because in wild-type females, there are clear, non-conflicting signals, while, in the experimental situation described here, autonomous male signals determine the fate of germ cells before female somatic signals are formed. Another alternative is that in somatic ovarian cells of wild-type females, feminizing signals are controlled not only by tra but also by Sxl. The hypothesis that somatic Sxl might be required for feminization of the germline has emerged before. XX germ cells that develop in an XY male will become spermatogenic. XX germ cells that develop in an XX male (e.g. genotype XX;tra/tra) become either spermatogenic or oogenic. The difference between the two types of males is that XX males have somatic Sxl activity. In fact, XX males lacking the somatic sex-determining function of Sxl have purely spermatogenic cells. These observations have led to the view that an early step of germline sex determination that occurs in the embryo might be followed by a second step: the production of a late feminizing signal could be controlled by the somatic function of Sxl, through a pathway that is unrelated to tra, tra-2, dsx and intersex. The idea that the feminizing signal should act late is based on the observation that XX;tra/tra gonads are initially male, that they appear to be feminized during larval stages and that nurse cells are differentiated during metamorphosis (Janzer, 2001 and references therein).

Although feminization of germ cells occurs late, it is somatic tra and not somatic Sxl that feminizes the germ cells. Feminizing tra function is sufficient to activate Sxl in germ cells, but tra function is not necessary for this activation, since in XX;tra/tra pseudomales, oogenic germ cells and Sxl activity can be detected. There must therefore be a certain redundance in the genetic control of feminizing signals, which ensures that the sex of germ cells is properly differentiated (Janzer, 2001 and references therein).

It is concluded that XX germ cells have been shown to adapt to their environment. In females they express female-specific genes and in males they express male-specific genes. Now it becomes clear that XY germ cells also respond to somatic signals. XX and XY germ cells differ in an initial phase during which XX germ cells adapt to the sex of the surrounding cells and XY germ cells autonomously enter the male pathway. After this initial phase, however, both types of germ cells develop in a non-autonomous way. In mammals, XX and XY germ cells do not behave differently early in development. Both types of germ cells are masculinized by somatic signals emanating from the male genital ridge from which the testis is differentiated in mice. In Drosophila, sex-determining signals do not only come from one tissue, the testis. Rather it seems that different sex-specific signals act in a complex and partly redundant way to control the expression of sex-specific genes in the germline. To understand how somatic signals derived either from the testis or from the ovary control gene expression within germ cells, it will be interesting to learn the nature of the molecules that form and transmit the sex-determining somatic signals (Janzer, 2001).

It will also be interesting to learn the molecular nature of the autonomous difference between XX and XY germ cells. It is known that the elements counting the X:A ratio of somatic cells do not perform a similar function in germ cells. Other elements must thus form the molecular basis of the germline X:A ratio, the autonomous signal that causes XX and XY germ cells to respond differently to somatic signals (Janzer, 2001).

Germ line regulation of Sxl

The genes sc, sis-a and runt needed to activate Sxl in the soma seem not to be required to activate this gene in the germ line; therefore, the X:A signal would composed of different genes in somatic and germ-line tissues (Granadino, 1993).

Alleles of the locus < I>ovarian tumor (otu) produce a phenotype known as ovarian tumors: ovarioles are filled with numerous poorly differentiated germ cells. These mutant germ cells have a morphology similar to primary spermatocytes, and they express male germ line-specific reporter genes. This indicates that they are engaged along the male pathway of germ line differentiation. Consistent with this conclusion, the splicing of Sex-lethal pre-mRNAs occurs in the male-specific mode in otu mutant transformed germ cells. The sexual transformation of the germ cells observed with several combinations of otu alleles can be reversed by constitutive expression of Sxl. This shows that otu acts upstream of Sxl in the process of germ line sex determination. The otu locus acts, along with ovo, snf, and Sxl, in a pathway (or parallel pathways) required for proper sex determination of the female germ line (Pauli, 1993).

The ovo locus encodes zinc finger transcription factor required for required upstream of splicing factors for female-specific splicing of Sex-lethal+ pre-mRNA. ovo reporter genes show high activity in the germ line of females and low activity in the germ line of males. XY flies transformed into somatic females do not show high levels of reporter activity, while XX flies transformed into somatic males do. This shows that high level ovo expression depends on the number of X chromosomes, not the somatic sexual signals. The requirement for ovo function is restricted to XX flies. Mutations in ovo have no effect on XY males, X0 males or XY females, but have pronounced effects on germ cell viability in XX females, XX females with sex transformed germ lines, and XX males indicating that ovo gene products are required for events occurring only in flies with two X chromosomes (Oliver, 1994).

The sex determination master switch, Sex-lethal, regulates the mitosis of early germ cells in Drosophila. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an immunoprecipitation experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of ornithine decarboxylase (ODC) antizyme, was isolated (Vied, 2003).

Mammalian Antizyme negatively regulates ODC catalytically as well by directing the inactivated enzyme to the proteasome for degradation. This negative regulation of ODC is part of a feedback loop that controls the levels of polyamines within the cell. Translation of Antizyme is dependent on ribosomal frameshifting, which is promoted by high levels of polyamines. As polyamine levels in the cell rise, more Antizyme is synthesized, leading to the turnover of ODC. Polyamines have been implicated in many processes, including cell growth, transcription, and differentiation. In mammals Antizyme and ubiquitin are thought to be respectively two types of proteasome targeting devices that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome (Vied, 2003 and references therein).

Drosophila gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. Gutfeeling appears to regulate the nuclear entry of Cyclin B as well. Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis (Vied, 2003).

Both forms of guf RNA are readily detected although the yield of mature RNA is greater than the precursor transcript in both wild-type and Sxlf4 ovaries. The overall yield from wild-type ovaries was lower for both products, but since the immunoprecipitations and RT-PCRs are not quantitative, this comparison is only speculative. To determine whether Sxl also binds guf RNA in somatic tissues, the immunoprecipitation and RT-PCR performed using embryonic extracts. Sxl also binds to spliced and unspliced guf RNA in embryos (ratio of spliced to unspliced is approximately equal). These data suggest that Sxl may regulate both the splicing and translation of guf (Vied, 2003).

Since Sxl binds to guf RNA in both the soma and germline, RT-PCR amplifications were performed for the four guf transcripts to determine whether any sex-specific products could be detected. guf RNA was analyzed from males and females (whole animals and carcasses) as well as testes and ovaries. For all cDNA types, no significant difference between the sexes was observed. From these data, it is concluded that Sxl does not regulate the alternative splicing of guf RNA in a global manner. Given the general requirement for guf, splicing regulation of guf RNA by Sxl may be restricted to only a subset of cells, such as the early germ cells, making detection of altered transcripts unlikely. Since there are no apparent alternative exons within guf, it is also possible that Sxl causes a retention of the introns leading to the degradation of the RNA. Alternatively, the regulation by Sxl may occur primarily at the level of translation control (Vied, 2003).

While the RNA binding data suggest that guf is a target of Sxl, a genetic interaction between the two genes would strengthen the idea that the two genes have a related function. Therefore the dose of guf was reduced in homozygous Sxlf4 females, which normally have small ovaries of tumorous egg chambers. This was done by using a P-element insertion allele (guf118-3) or a small deletion allele produced by the imprecise excision of the guf118-3 P-element (guflex47; Salzberg, 1996). Reducing guf dose in a homozygous Sxlf4 background rescues the Sxlf4 phenotype. The females lay eggs that hatch and produce adults (Vied, 2003).

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes a stable heterodimer [METTL3 (methyltransferase-like 3) and METTL14], WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 (Inducer of meiosis 4) , Mettl14 (Methyltransferase-like 14 ), the Wilms tumour 1-associated protein Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. This study shows that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout model, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression (Haussmann, 2016).

In mature mRNA the m6A modification is most prevalently found around the stop codon as well as in 5' untranslated regions (UTRs) and in long exons in mammals, plants and yeast. Since methylosome components predominantly localize to the nucleus, it has been speculated that m6A localized in pre-mRNA introns could have a role in alternative splicing regulation in addition to such a role when present in long exons. This prompted an investigation of whether m6A is required for Sxl alternative splicing, which determines female sex and prevents dosage compensation in females. A null allele of the Drosophila METTL3 methyltransferase homologue Ime4 was induced by imprecise excision of a P element inserted in the promoter region. The excision allele Δ22-3 deletes most of the protein-coding region, including the catalytic domain, and is thus referred to as Ime4null. These flies are viable and fertile, but both flightless and this phenotype can be rescued by a genomic construct restoring Ime4. Ime4 shows increased expression in the brain and, as in mammals and plants, localizes to the nucleus (Haussmann, 2016).

Following RNase T1 digestion and 32P end-labelling of RNA fragments, m6A was detected after guanosine (G) in poly(A) mRNA of adult flies at relatively low levels compared to other eukaryotes, but at higher levels in unfertilized eggs. After enrichment with an anti-m6A antibody, m6A is readily detected in poly(A) mRNA, but absent from Ime4null flies (Haussmann, 2016).

As found in other systems, and consistent with a potential role in translational regulation, m6A was detected in polysomal mRNA, but not in the poly(A)-depleted rRNA fraction. This also confirmed that any m6A modification in rRNA is not after G in Drosophila (Haussmann, 2016).

Consistent with the hypothesis that m6A plays a role in sex determination and dosage compensation, the number of Ime4null females was reduced to 60% compared to the number of males, whereas in the control strain female viability was 89%. The key regulator of sex determination in Drosophila is the RNA-binding protein Sxl, which is specifically expressed in females. Sxl positively auto-regulates expression of itself and its target transformer (tra) through alternative splicing to direct female differentiation. In addition, Sxl suppresses translation of msl-2 to prevent upregulation of transcription on the X chromosome for dosage compensation; full suppression also requires maternal factors. Accordingly, female viability was reduced to 13% by removal of maternal m6A together with zygotic heterozygosity for Sxl and Ime4 (Ime4Δ22-3 females crossed with Sxl7B0 males, a Sxl null allele). Female viability of this genotype is completely rescued by a genomic construct or by preventing ectopic activation of dosage compensation by removal of msl-2. Hence, females are non-viable owing to insufficient suppression of msl-2 expression, resulting in upregulation of gene expression on the X chromosome from reduced Sxl levels. In the absence of msl-2, disruption of Sxl alternative splicing resulted in females with sexual transformations displaying male-specific features such as sex combs, which were mosaic to various degrees, indicating that Sxl threshold levels are affected early during establishment of sexual identities of cells and/or their lineages. In the presence of maternal Ime4, Sxl and Ime4 do not genetically interact (Sxl7B0/FM7 females crossed with Ime4null males, 103% female viability. In addition, Sxl is required for germline differentiation in females and its absence results in tumorous ovaries. Consistent with this, tumorous ovaries in Sxl7B0/+;Ime4null/+ daughters from Ime4null females or heterozygous Sxl7B0 females (Haussmann, 2016).

Furthermore, levels of the Sxl female-specific splice form were reduced to approximately 50%, consistent with a role for m6A in Sxl alternative splicing . As a result, female-specific splice forms of tra and msl-2 were also significantly reduced in adult females (Haussmann, 2016).

To obtain more comprehensive insights into Sxl alternative splicing defects in Ime4null females, splice junction reads were examined from RNA-seq. Besides the significant increase in inclusion of the male-specific Sxl exon in Ime4null females, cryptic splice sites and increased numbers of intronic reads were detected in the regulated intron. Consistent with reverse transcription polymerase chain reaction (RT–PCR) analysis of tra, the reduction of female splicing in the RNA sequencing is modest, and as a consequence, alternative splicing differences of Tra targets dsx and fru were not detected in whole flies, suggesting that cell-type-specific fine-tuning is required to generate splicing robustness rather than being an obligatory regulator. In agreement with dosage-compensation defects as a main consequence of Sxl dysregulation in Ime4null mutants, X-linked, but not autosomal, genes are significantly upregulated in Ime4null females compared to controls (Haussmann, 2016).

Furthermore, Sxl mRNA is enriched in pull-downs with an m6A antibody compared to m6A-deficient yeast mRNA added for quantification. This enrichment is comparable to what was observed for m6A-pull-down from yeast mRNA (Haussmann, 2016).

To map m6A sites in the intron of Sxl, an in vitro m6A methylation assay was employed using Drosophila nuclear extracts and labelled substrate RNA. m6A methylation activity was detected in the vicinity of alternatively spliced exons. Further fine-mapping localized m6A in RNAs C and E to the proximity of Sxl-binding sites. Likewise, the female-lethal single amino acid substitution alleles fl(2)d1 and vir2F interfere with Sxl recruitment, resulting in impaired Sxl auto-regulation and inclusion of the male-specific exon. Female lethality of these alleles can be rescued by Ime4null heterozygosity, further demonstrating the involvement of the m6A methylosome in Sxl alternative splicing (Haussmann, 2016).

Next, alternative splicing changes was globally analyzed in Ime4null females compared to the wild-type control strain. A statistically significant reduction in female-specific alternative splicing of Sxl was observed. In addition, 243 alternative splicing events in 163 genes were significantly different in Ime4null females, equivalent to around 2% of alternatively spliced genes in Drosophila. Six genes for which the alternative splicing products could be distinguished on agarose gels were confirmed by RT-PCR. Notably, lack of Ime4 did not affect global alternative splicing and no specific type of alternative splicing event was preferentially affected. However, alternative first exon (18% versus 33%) and mutually exclusive exon (2% versus 15%) events were reduced in Ime4null compared to a global breakdown of alternative splicing in wild-type Drosophila, mostly to the extent of retained introns (16% versus 6%), alternative donor (16% versus 9%) and unclassified events (14% versus 6%). Notably, the majority of affected alternative splicing events in Ime4null were located to the 5' UTR, and these genes had a significantly higher number of AUG start codons in their 5' UTR compared to the 5' UTRs of all genes. Such a feature has been shown to be relevant to translational control under stress conditions. (Haussmann, 2016 and references therein).

The majority of the 163 differentially alternatively spliced genes in Ime4 females are broadly expressed (59%), while most of the remainder are expressed in the nervous system (33%), consistent with higher expression of Ime4 in this tissue. Accordingly, Gene Ontology analysis revealed a highly significant enrichment for genes involved synaptic transmission (Haussmann, 2016).

Since the absence of m6A affects alternative splicing, m6A marks are probably deposited co-transcriptionally before splicing. Co-staining of polytene chromosomes with antibodies against haemagglutinin (HA)-tagged Ime4 and RNA Pol II revealed broad co-localization of Ime4 with sites of transcription, but not with condensed chromatin-visualized with antibodies against histone H4. Furthermore, localization of Ime4 to sites of transcription is RNA-dependent, as staining for Ime4, but not for RNA Pol II, was reduced in an RNase-dependent manner (Haussmann, 2016).

Although m6A levels after G are low in Drosophila compared to other eukaryotes, broad co-localization of Ime4 to sites of transcription suggests profound effects on the gene expression landscape. Indeed, differential gene expression analysis revealed 408 differentially expressed genes where 234 genes were significantly upregulated and 174 significantly downregulated in neuron-enriched head/thorax of adult Ime4null females. Cataloguing these genes according to function reveals prominent effects on gene networks involved in metabolism, including reduced expression of 17 genes involved in oxidative phosphorylation. Notably, overexpression of the m6A mRNA demethylase FTO in mice leads to an imbalance in energy metabolism resulting in obesity (Haussmann, 2016).

Next, tests were performed to see whether either of the two substantially divergent YTH proteins, YT521-B and CG6422, decodes m6A marks in Sxl mRNA. When transiently transfected into male S2 cells, YT521-B localizes to the nucleus, whereas CG6422 is cytoplasmic. Nuclear YT521-B can switch Sxl alternative splicing to the female mode and also binds to the Sxl intron in S2 cells. In vitro binding assays with the YTH domain of YT521-B demonstrate increased binding of m6A-containing RNA. In vivo, YT521-B also localizes to the sites of transcription (Haussmann, 2016).

To further examine the role of YT521-B in decoding m6A Drosophila strain YT521-BMI02006 was analyzed, where a transposon in the first intron disrupts YT521-B. This allele is also viable, and phenocopies the flightless phenotype and the female Sxl splicing defect of Ime4null flies. Likewise, removal of maternal YT521-B together with zygotic heterozygosity for Sxl and YT521-B reduces female viability and results in sexual transformations such as male abdominal pigmentation. In addition, overexpression of YT521-B results in male lethality, which can be rescued by removal of Ime4, further reiterating the role of m6A in Sxl alternative splicing. Since YT521-B phenocopies Ime4 for Sxl splicing regulation, it is the main nuclear factor for decoding m6A present in the proximity of the Sxl-binding sites. YT521-B bound to m6A assists Sxl in repressing inclusion of the male-specific exon, thus providing robustness to this vital gene regulatory switch (Haussmann, 2016).

Nuclear localization of m6A methylosome components suggested a role for this 'fifth' nucleotide in alternative splicing regulation. The discovery of the requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing has important implications for understanding the fundamental biological function of this enigmatic mRNA modification. Its key role in providing robustness to Sxl alternative splicing to prevent ectopic dosage compensation and female lethality, together with localization of the core methylosome component Ime4 to sites of transcription, indicates that the m6A modification is part of an ancient, yet unexplored mechanism to adjust gene expression. Hence, the recently reported role of m6A methylosome components in human dosage compensation further support such a role and suggests that m6A-mediated adjustment of gene expression might be a key step to allow for the development of the diverse sex determination mechanisms found in nature (Haussmann, 2016).

Factors influencing somatic sex characteristics

The doublesex (dsx) locus encodes male-specific and female-specific transcription factors that are essential for the proper differentiation of sexually dimorphic somatic features of Drosophila melanogaster. The female specific polypeptide is created by Sex-lethal splicing of DSX mRNA. Ectopic expression of the male-specific DSX polypeptide in either males or females results in three novel phenotypes: transformation of bristles on all legs toward a sex comb-like morphology, pigmentation of dorsal spinules and ventral setae in third-instar larvae, and lethality. These results provide evidence that the role of the male DSX protein includes activation of some aspects of male differentiation as well as repression of female differentiation (Jursnich, 1993).

The two different Doublesex proteins have an additional role in regulation of transcription of Yp1 and Yp2, two linked jointly regulated genes expressed in the adult fat body. Sex specificity in fat bodies arises from opposing effects of the male DSX and femal DSX splice variants. The female Doublesex splice varient activates Yp1 and Yp2 by sterically excluding a repressor (AEF1) from the promoter and acting synergistically with an activator (bzip1). The male splice varient acts as a repressor at the same site. Similar regulation occurs in ovarian somatic cells. Thus Doublesex regulates male and female characteristics depending on which splice varient is produced in the two sexes (An, 1995).

Post-splicing regulation of Sxl

The Sex-lethal (Sxl) gene is required in Drosophila females for sexual differentiation of the soma, for germ cell differentiation and dosage compensation. Three new alleles of female-lethal-on-X (flex), an X-linked female-lethal mutation, have been isolated, characterized, and flex function in sex determination has been examined. Sxl protein is missing in flex/flex embryos, however transcription from both SxlPe, the early Sxl promoter and SxlPm, the late maintenance promoter, is normal in flex homozygotes. In flex/flex embryos, SXL mRNA is spliced in the male mode. Analysis of flex germline clones shows that it also functions in oogenesis, but in contrast to Sxl mutants that show an early arrest tumorous phenotype, flex mutant egg chambers develop to stage 10. In flex ovarian clones, SXL mRNA is also spliced in the male form. Hence, flex is a sex-specific regulator of Sxl functioning in both the soma and the germline. Genetic interaction studies show that flex does not enhance female lethality of Sxl loss-of-function alleles but it rescues the male-specific lethality of both of the gain-of-function Sxl mutations, SxlM1 and SxlM4. In contrast to mutations in splicing regulators of Sxl, the female lethality of flex is not rescued by either SxlM1 or SxlM4. Based on these observations, it is proposed that flex regulates Sxl at a post-splicing stage and regulates either its translation or the stability of the Sxl protein (Bhattacharya, 1999).

Sxl msl2 splicing

The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Splicing inhibition of msl-2 requires a binding site for Sxl at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by Sxl. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that Sxl can no longer displace it from the poly(Y) tract. These results reveal a novel function for U2AF35, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35-AG interaction to regulate intron removal (Merendino, 1999).

The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 transcripts. Sxl blocks the binding of U2 auxiliary factor (U2AF see U2 small nuclear riboprotein auxiliary factor 50) to the polypyrimidine (Py)-tract associated with the 3' splice site of the regulated intron. This study reports that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5' splice site is required for efficient splicing inhibition by Sxl. Psoralen-mediated crosslinking experiments suggest that Sxl binding to this uridine-rich sequence inhibits recognition of the 5' splice site by U1 snRNP in HeLa nuclear extracts. Sxl interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5' splice site and for splicing of this pre-mRNA, it is proposed that Sxl antagonizes TIA-1 activity and thus prevents 5' splice site recognition by U1 snRNP. Taken together with previous data, it is concluded that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by Sxl (Forch, 2001).

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires Sex-lethal (Sxl) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. The msl-2 mRNA sequence elements that are important for regulation by Sxl have been delineated and functionally critical sequences have been identified adjacent to regulatory Sxl binding sites. Sxl inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster (Gebauer, 2003).

Following the delineation of the mRNA elements that contribute to or are dispensable for the control of Msl-2 synthesis by Sxl, a 'minimal mRNA' (WTS) was generated that recapitulates the critical features of msl-2 mRNA regulation. The stepwise substitution of the 3992 nt msl-2 mRNA by the much shorter WTS mRNA (336 nt) proved critical to the success of the ribosome assembly assays because only this smaller mRNA yields a sufficiently high resolution between the different translation initiation intermediates in sucrose gradient assays (Gebauer, 2003).

The specific inhibition of 80S ribosomal complex formation in the presence of the translation elongation inhibitor cycloheximide shows that Sxl inhibits the initiation step of translation. This result does not exclude the possibility that an additional postinitiation step could also be affected. Importantly, the assembly of early initiation intermediates carrying only the small ribosomal subunit is clearly inhibited by Sxl. This observation is consistent with two possible scenarios: the regulatory mechanism may inhibit the initial, cap-mediated recruitment of the small ribosomal subunit to the mRNA, or it may interfere with the scanning of a recruited 43S complex to the translation initiation codon. The latter mechanism could prevent the stable association of the 43S complex with the mRNA because scanning intermediates are thought to readily dissociate from the mRNA (Gebauer, 2003).

Sxl must bind to both the 5' and 3' UTRs of msl-2 mRNA to inhibit translation efficiently. This requirement distinguishes Sxl from most other regulators, which inhibit translation by binding to only one of the untranslated regions, commonly the 3' UTR. The 3' UTR of msl-2 contributes a function in addition to Sxl binding. The critical sequence elements are located in close proximity to the 3' UTR Sxl binding sites E and F. It is suggested that Sxl and the regulatory sequences flanking its 3' UTR binding sites may cooperate in the recruitment of translational corepressors (Gebauer, 2003).

Both Sxl and the iron regulatory proteins (IRP) inhibit the stable association of the 40S ribosomal subunit with their target mRNAs. However, the regulatory mechanisms used by IRP and Sxl differ in several aspects. In contrast to Sxl, IRP must bind to a cap-proximal hairpin in ferritin mRNA to effectively block the binding of the 43S complex to the mRNA. Moreover, the IRP mechanism does not require contributions from the 3' UTR, as does Sxl. Furthermore, Sxl binding is not sufficient for translation inhibition of msl-2 mRNA, and a simple steric hindrance mechanism is hence unlikely to explain Sxl function. By contrast, the mere occupancy of a cap-proximal hairpin by a high-affinity RNA binding protein suffices for the IRP mechanism of translational control (Gebauer, 2003).

Translational repression of msl-2 mRNA by Sxl is independent of the presence of a cap structure and a poly(A) tail. This feature differs from other translational regulators such as CPEB. Because both the cap structure and the poly(A) tail play critical roles in promoting the recruitment of the small ribosomal subunit to mRNAs, these findings raise the interesting question of how Sxl regulates the interaction between the mRNA and the small ribosomal subunit independently of both of these mRNA end modifications. An intriguing possibility is that Sxl targets the scanning step of translation rather than the initial recruitment of the 43S complex. Translation inhibition may occur by formation of a higher order complex of repressors bound to the 5' and 3' UTRs of msl-2 mRNA. It is also possible that Sxl/corepressor interactions create a regulatory surface that targets specific translation initiation factors. Future experiments will aim to identify the responsible components and molecular interactions (Gebauer, 2003).

Drosophila Sex-lethal-mediated translational repression of male-specific lethal 2 (msl-2) mRNA is essential for X-chromosome dosage compensation. Binding of Sxl to specific sites in both untranslated regions of msl-2 mRNA is necessary for inhibition of translation initiation. This study describes the organization of Sxl as a translational regulator and shows that the RNA binding and translational repressor functions are contained within the two RRM domains and a C-terminal heptapeptide extension. The repressor function is dormant unless Sxl binds to msl-2 mRNA with its own RRMs, because Sxl tethering via a heterologous RNA-binding peptide does not elicit translational inhibition. This study reveals proteins that crosslink to the msl-2 3' untranslated region (3'UTR) and co-immunoprecipitate with Sxl in a fashion that requires its intact repressor domain and correlates with translational regulation. Translation competition and UV-crosslink experiments show that the 3'UTR msl-2 sequences adjacent to Sxl-binding sites are necessary to recruit titratable co-repressors. These data support a model where Sxl binding to the 3'UTR of msl-2 mRNA activates the translational repressor domain, thereby enabling it to recruit co-repressors in a specific fashion (Grsckovic, 2003).

The architecture and function of Sxl as a translational repressor was analyzed. In female flies, translational repression of msl-2 mRNA by Sxl represents a critical regulatory step for dosage compensation. The results discriminate Sxl's function as a splicing regulator from its function in translational control. They also suggest that the translational repressor activity of Sxl is dormant, and activated upon binding to the msl-2 mRNA. Finally, functional evidence is provided that Sxl and the msl-2 3'UTR cooperate in the recruitment of proteins that appear to act as translational co-repressors (Grsckovic, 2003).

Modularity represents one of the most common principles of protein architecture. It permits the organization of biological functions into independent domains, and integration of these functions in a single molecule. This work shows that the mRNA binding and translational regulatory functions of Sxl are not organized into independent domains. Rather, both functions reside within aa 122-301 of the protein, a region that has been recognized as the RNA-binding domain of Sxl. The N-terminal domain of Sxl contributes an essential function only to alternative splicing, but is dispensable for translational control of msl-2 mRNA. The embedding of the translational regulatory function into the RNA-binding domain and a few additional C-terminal amino acids also explains why N-terminal truncations strongly impair Sxl function in sex determination, but not in dosage compensation in vivo (Grsckovic, 2003).

Based on the analysis of hybrid proteins between Drosophila and Musca Sxl, it is suggested that the region corresponding to RRM1 (aa 122-200) makes an important contribution to translational repression and represents a critical difference between Drosophila Sxl, which efficiently represses translation, and Musca Sxl, which does not. In addition to RRM1, an extension of 7 aa following the RRMs cooperates in translation inhibition. Interestingly, RRM1 has been shown to contact the snRNP component SNF, a protein thought to be involved in the autoregulatory splicing of Sxl pre-mRNA. RRM1 also binds SIN, a protein of unknown function. However, both SNF and SIN are much smaller than the crosslinked polypeptides that Sxl recruits to the 3'UTR of msl-2. The crystal structure of the Sxl RRMs bound to the RNA reveals that almost all of the residues that differ from those in mSXL are exposed on the accessible outer surface of the protein. None of them contacts the RNA or other parts of the RRMs. In accordance with these structural data, similar RNA-binding affinities of dRBD3 and mRBD are found in gel mobility-shift assays. Exposed amino acid residues on the outer surface of the RNA-binding domain of Sxl are ideally placed to mediate the interaction of Sxl with translational co-repressors (Grsckovic, 2003).

Regulatory proteins that bind to specific mRNAs to control their translation employ different modes of action. In all cases, specificity demands that the regulatory protein should affect the translation of the mRNAs to which it binds, but not perturb the general translation machinery in a non-specific way. One solution to this problem is steric control, where the mRNA binding event is both necessary and sufficient for translational repression. In these cases, no translational effector domains per se are required, which could potentially act in trans and interfere with the function of a translation factor while the regulatory protein is not bound to its target mRNA. Steric control has been shown for the iron regulatory proteins, and is also evident in the translational inhibition of the bacteriophage lambda -- the lambda anti-terminator protein. msl-2 mRNA regulation by Sxl does not operate by such a steric mechanism, and the specificity problem must be solved differently (Grsckovic, 2003).

The binding of Sxl to its physiological binding sites in the 5' and 3'UTRs of WT or BLEF mRNA (a construct that harbours all msl-2 mRNA sequences required for full regulation by Sxl) cannot be substituted by tethered Sxl, suggesting that Sxl must bind the RNA with its own RRMs to be active as a translational repressor. This contrasts with other RNA-binding proteins, such as the poly(A)-binding protein (PABP) or proteins involved in nonsense-mediated mRNA decay, which retain their respective functions when being tethered to an mRNA. Importantly, the lambdaSXL protein (a hybrid protein containing the RNA-binding domain of the lambda phage anti-terminator protein, a 22 aa oligomer referred to as the lambda peptide) binds to the indicator mRNAs, as evidenced by its steric regulatory function and gel mobility-shift assays, and it is fully functional as a bona fide msl-2 regulatory protein when binding through its own RRMs. This excludes trivial misfolding effects as an explanation for the lack of tethered function (Grsckovic, 2003).

It seems most likely that the dormant translational repressor domain, which is embedded within the RNA-binding region of Sxl, is only activated when the protein binds to msl-2 mRNA, solving the specificity problem. Sxl binding to the EF region of the msl-2 3'UTR induces the binding of additional proteins to this site, which cannot be crosslinked in the absence of Sxl. It is possible that two molecules of Sxl binding to the adjacent E and F sites, respectively, cooperate in recruiting the co-repressor(s) by creating a binding surface partially lacking from monomeric Sxl. The recruitment of these additional proteins has two requirements. (1) Sxl but not musca SXL can mediate the crosslinking of these factors. This suggests that specific amino acids that are exposed on Sxl (or Sxl molecules binding closely to each other) contribute to the interaction. (2) Sxl binding to the 3'UTR probe but not the 5'UTR or mutated 3'UTR probes is required for crosslinking. This suggests that the 3'UTR RNA sequences make an additional specific contribution. Indeed, in addition to Sxl-binding sites E and F, sequences adjacent to these sites are essential for recruitment of the additional proteins. These factors that bind to the 3'UTR regulatory region are functional co-repressors, because only the 3'UTR WT RNA competes for translational inhibition, while the mut2456 and 5' RNAs (which bind Sxl but not the co-repressors) fail to do so (Grsckovic, 2003).

The proposed model for Sxl bears interesting parallels with the function of Pumilio (Pum) and other members of the Puf family of translational regulators. Pum inhibits the translation of maternal hunchback mRNA as part of a complex that is sequentially built on the Nanos-response element (NRE) in the 3'UTR of the message, and the translational repressor domain of Pum is embedded within its RNA binding region, the so-called Puf repeat. Binding of Pum to the NRE triggers the recruitment of Nanos and this event, in turn, stimulates the binding of Brain Tumor. Hence, the stepwise assembly of co-repressor complexes, which requires multiple specific protein-protein and RNA-protein interactions, emerges as a theme in translational control. The msl-2 regulatory complex inhibits translation initiation by blocking the stable binding of the small ribosomal subunit to the mRNA in a cap-independent fashion. Future work will aim to define the composition of this regulatory complex and reveal its molecular interactions with the machinery that executes early steps in translation initiation (Grsckovic, 2003).

Drosophila MSL-2 is the limiting component of the dosage compensation complex (DCC). Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (Sxl) binding to sites in both the 5' and the 3' untranslated regions (UTRs). This study uncovers the mechanism by which Sxl achieves tight control of translation initiation. Sxl binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by Sxl bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA (Beckmann, 2005 ).

This study investigates how Sxl silences translation of the msl-2 mRNA. In female flies, this regulation prevents the formation of the dosage compensation complex and thus deleterious hypertranscription of the two female X chromosomes. The results define the molecular basis of this critical regulatory step. They also reveal a mechanism of translational control that is based on the integration of two separable components. Individually, each of the two components provides insights into functional properties of 5′ and 3′ regulatory complexes to interfere with translation initiation, and each of these two components appears to be mechanistically unprecedented (Beckmann, 2005).

In male flies, MSL-1 and MSL-2 mediate the assembly of the DCC on the single X chromosome, which is thought to spread along the entire chromosome promoting an ~2-fold increase in transcription levels. The absence of MSL-2 in females does not allow DCC formation, while transgenic female flies expressing MSL-2 assemble the complex, showing that MSL-2 is the limiting subunit of the DCC. Although the msl-2 transcript levels in females are reduced to 20%-30% of those in male flies, translational control mediated by Sxl is crucial to block MSL-2 expression (Beckmann, 2005).

The msl-2 mRNA contains two SXL binding sites within the 5'UTR and four binding sites within the 3'UTR (sites A-F). Sxl bound to either UTR of msl-2 mRNA inhibits translation by interfering with initiation prior to 48S complex formation. Furthermore, the two regulatory regions independently interfere with translation initiation by different means. Earlier evidence suggested that the roles of Sxl bound to the 5′UTR and the 3′UTR, respectively, are noninterchangeable: (1) the 5′UTR Sxl binding site B cannot substitute for sites E and F when introduced into the 3′UTR; (2) UV-crosslinking experiments identified at least one protein that is recruited by Sxl specifically to the 3′UTR (Grskovic, 2003). The crosslinks require the sequences that flank the E and F sites, and RNA competition experiments functionally implicated this 3′UTR binding protein in Sxl-mediated translational control; by contrast, crosslinks to the 5′UTR are limited to Sxl itself. The simplest interpretation of this earlier work was that both UTRs engage in a functional and perhaps physical interaction to block the stable engagement of the small ribosomal subunit with the mRNA at a single step (Beckmann, 2005).

This work now reveals that such a simple assumption is incorrect. Rather than forming a single repressor complex that targets one step in the initiation pathway, Sxl acts as a bifunctional regulator: 3′UTR bound Sxl inhibits translation from cap-proximal and cap-distal AUGs identically well, while 5′UTR bound Sxl can only function when it binds upstream of the initiation codon. Based on these data and direct physical evidence provided by the toeprint experiments (to identify translation complexes that bind to the mRNA), it is concluded that Sxl bound to the 3′UTR impedes the initial recruitment of 43S complexes to the mRNA while 5′UTR bound Sxl stalls scanning 43S complexes upstream of its binding site (Beckmann, 2005).

How does Sxl binding to the B site achieve a scanning block? Apparently, Sxl hinders the transit of 43S complexes across site B. Steric hindrance of ribosomal scanning has been proposed for iron-regulatory proteins/iron-responsive element complexes that were introduced ~100 nucleotides downstream from the cap structure of a reporter mRNA. However, scanning inhibition by Sxl bound to the B site does not appear to follow a simple steric mechanism, i.e., to be imposed solely by high affinity mRNA binding. mRBD a Musca domestica Sxl derivative (Musca SXL does not function in X chromosome dosage compensation and mRBD does not repress msl-2) fails to repress translation or stall scanning 43S complexes, although it binds to site B with an affinity as high as that of Sxl. Furthermore, tethering of a λ peptide-Sxl fusion protein to a BoxB element replacing Sxl binding site B in the 5′UTR of msl-2 mRNA does not inhibit translation despite the high affinity of the λ peptide-BoxB interaction (Beckmann, 2005).

Therefore, it is proposed that Sxl regulates scanning either by altering the 5′UTR secondary structure and/or promoting the formation of a higher-order assembly on the B site. Such a complex could then act as a (steric) roadblock to scanning, without being able to halt elongating 80S ribosomes. Alternatively, Sxl and potential interacting proteins may specifically interfere with the function of a translation initiation factor or the small ribosomal subunit which is required for scanning but not for translation elongation. Interestingly, site B is composed of 16 uridine residues that could be bound by a Sxl dimer. Since Sxl engages in protein-protein interactions through its RNA binding domains, Sxl dimerization and additional factors recruited by Sxl may promote the formation of a higher-order repressor complex that blocks scanning, in as much as a stalled elongating ribosome can function as a block to scanning (Beckmann, 2005).

How does Sxl bound to the 3′UTR inhibit 43S preinitiation complex recruitment? The recruitment of the 43S complex represents a previously identified target of 3′UTR binding translational regulators. For example, Maskin, Cup, and Bicoid block the recruitment of the 43S complex by interfering with the assembly of eIF4F at the cap structure. However, Sxl-mediated inhibition is independent of the cap structure (Gebauer, 2003), implying that the 43S complex recruitment block imposed by Sxl is different from that mediated by these regulators (Beckmann, 2005).

It is noticed that 3′UTR-mediated translational inhibition by Sxl involves the accumulation of the repressed mRNA within unusually heavy RNP particles. Interestingly, Sxl has previously been found in large, RNase-sensitive complexes sedimenting faster than bulk mRNPs in sucrose density gradient experiments. An attractive possibility is that Sxl in association with the 3′UTR corepressor nucleates a large repressor complex or that the 3′UTR repressor complex promotes the multimerization of mRNPs leading to the formation of mRNP clusters. Multimerization of mRNPs has been observed during the localization of translationally silent bicoid mRNA to the anterior pole of the Drosophila oocyte. Clustered mRNAs may be less accessible to the translation initiation machinery, providing a possible mechanism of 3′UTR-mediated inhibition of 43S complex recruitment independent of mRNA-specific 3′ to 5′ end communication (Beckmann, 2005).

What are the biological advantages of the duality of translational control by Sxl? Such an integrated failsafe mechanism allows efficient repression of translation in situations where the leakiness of a single mechanism could be deleterious for the cell and/or the organism. Indeed, forced expression of the MSL-2 protein in female flies enables the loading of the dosage compensation complex onto the two X chromosomes and causes lethality. Therefore, the translational repression of female msl-2 mRNA must be robust, which is achieved by the combination of the two mechanisms that cooperate to prevent 43S complexes from reaching the initiation codon (Beckmann, 2005).

oskar (osk) mRNA translation in Drosophila oocytes also appears to be regulated at multiple steps. The synthesis of the posterior determinant Osk must be strictly restricted to the posterior pole of the embryo. This is achieved by the posterior accumulation of osk mRNA and the translational repression of unlocalized osk mRNA. The protein Bruno binds to Bruno-response elements (BREs) in the 3′UTR of the osk mRNA and is important for inhibition of translation. The fact that Bruno interacts with the repressor Cup suggested that the responsible mechanism targets translation initiation, although this has not been shown directly. A recent report identified a significant fraction of unlocalized osk mRNA in association with ribosomes, indicating that translation of the osk mRNA may be regulated (in addition) at a postinitiation step. Similar observations implicating multiple levels of translational control have also been made for the spatially and temporally controlled nanos mRNA in Drosophila embryos. In this case, both mechanisms may not be simultaneously active at all stages of development. Translational failsafe mechanisms like the one described here may become recognized as a more widespread principle of robust control over protein synthesis (Beckmann, 2005).

Sxl is in a complex that contains all of the known Hh cytoplasmic components: Hh promotes the entry of Sxl into the nucleus in the wing disc

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci (Horabin, 2003).

Sxl co-immunoprecipitates with Cos2 and Fu in the female germline. Since Ci is not expressed in germ cells, it is probable that a different Hh cytoplasmic complex might exist in germ cells. In somatic cells, Sxl is expressed in all female cells while Ci is expressed in only a subset. To test whether the Hh pathway differentiates between the two proteins in somatic cells, Sxl was immunoprecipitated from embryonic extracts and the immunoprecipitates probed for the various Hh cytoplasmic components. The immunoprecipitates showed that Cos2, Fu and Ci are complexed with Sxl. The specificity of this association of Sxl with the Hh pathway components was verified using antibodies to either Ci or Su(fu), and testing the immunoprecipitates for the presence of Sxl. Both co-immunoprecipitated with Sxl. The Ci immunoprecipitate was also tested for another Hh cytoplasmic component, Fu, which was present as expected. These interactions are maintained in a Su(fu)LP background (protein null allele). An IP of Ci from Su(fu)LP embryos brought down Sxl, as well as Fu and Cos2. Taken together, these data suggest that cells that express Ci and Sxl have both proteins in the same complex with the known cytoplasmic components of the Hh signaling pathway (Horabin, 2003).

Previous work on the germline has suggested that the Hh signaling pathway affects the intracellular trafficking Sxl. The cross talk between these two developmental pathways has been analyzed in tissues where both Hh targets can be present in the same cell. While analysis of embryos only uncovered an effect of Cos2 on Sxl, analysis of wing discs allowed several specific effects to be uncovered. At least three new functional aspects of the Hh pathway are suggested:

  1. More than one 'target' protein can exist in the Hh cytoplasmic complex.
    Immunoprecipitation experiments using extracts from embryos indicate that Sex-lethal and the known Hh signaling target Ci are in the same complex. The two proteins can co-immunoprecipitate each other as well as other known members of the Hh cytoplasmic complex. Even when Su(fu), the cytoplasmic component that most strongly anchors Sxl in the cytoplasm, is removed, Sxl can still be co-immunoprecipitated with both Ci and Fu. As a whole, these results suggest that at least some proportion of the two Hh 'target' proteins are in a common complex within the cell. Additionally, the wing defects produced when Sxl is overexpressed in the Hh signaling region suggest that their relative concentrations are important for their normal functioning (Horabin, 2003).
  2. The Hh targets can be affected differentially.
    The presence of two 'targets' within the Hh cytoplasmic complex, raises the question of how they can be differentially affected. The data show that the various members of the Hh pathway do not affect Sx1 and Ci similarly. Smo appears to be dispensable for the transmission of the Hh signal in promoting Sx1 nuclear entry, while Smo is critical for the activation of Ci. Conversely, while Ptc is essential for the effect of Hh on Sxl, it is dispensable for the activation of Ci. The Fu kinase (fumH63 background) also appears to have no role in Hh signaling with respect to Sxl, while it is critical for the activation of Ci. By contrast, both Su(fu) and the Fu regulatory domain act similarly on Sxl and Ci, serving to anchor them in the cytoplasm (Horabin, 2003).

    Taken together, these data suggest that the presence of Hh can be relayed to the cytoplasmic components differentially and, while the data do not address the point, suggest how different outcomes might be achieved. Ptc has been proposed to be a transmembrane transporter protein that functions catalytically in the inhibition of Smo via a diffusible small molecule. The stimulation of Sxl nuclear entry by the binding of Hh to Ptc might also involve a change in the internal cell milieu, but in this case the Hh cytoplasmic complex may be affected independently, not requiring a change in the activity of Smo or the Fu kinase (Horabin, 2003).

  3. Ptc can signal the presence of the Hh ligand in a positive manner.
    Several experiments indicate that Hh bound to Ptc enhances the nuclear entry of Sxl. That Smo has no role in transmitting the Hh signal is most clearly demonstrated by expressing the PtcD584 protein in both the anterior and posterior compartments of the dorsal half of the wing disc. PtcD584 acts as a dominant negative and so activates Ci in the anterior compartment, but it fails to enhance the levels of nuclear Sxl in the anterior because it sequesters Hh in the posterior compartment. The double mutant condition of ptc clones in a hhMRT background clearly places Ptc downstream of Hh, while showing Ptc can act positively in transmitting the Hh signal (Horabin, 2003).

A positive role for Ptc, but in this case in conjunction with Smo, in promoting cell proliferation during head development has recently been reported. In this situation, however, Hh acts negatively on both Ptc and Smo in their activation of the Activin type I receptor, suggesting an even greater variance from the canonical Hh signaling process (Horabin, 2003).

While the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Su(fu) is expressed uniformly across the disc so it does not appear to be responsible for the AP differences, and ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment). Removal of Hh, however, reduces the nuclear entry rate of Sxl in both compartments. In this regard, the parallel between Hh pathway activation and Sxl nuclear entry in the posterior compartment is worth noting. Fu is also activated in the posterior compartment in a Hh-dependent manner, even though Ptc is not present. It is not clear what mediates between Hh and Fu (Horabin, 2003).

The data also suggest that the Hh cytoplasmic complex may have slightly different compositions in different tissues and/or at developmental stages. In the female germline and in embryos, the absence of Cos2 leads to a severe reduction in Sxl levels. However, in wing discs when mutant clones are made using the same cos2 allele, there is no effect on Sxl. It is suggested that between the third instar larval stage and eclosion, the composition of the Hh cytoplasmic complex may change again to make Sxl more sensitive to Cos2. This would explain why removal of Cos2 can produce sex transformations of the foreleg even though mutant clones in wing discs (and also leg discs) show no alterations in Sxl levels (Horabin, 2003).

A similar argument might apply to the weak sex transformations of forelegs produced by PKA clones. Alternatively, PKA may have a very weak effect but the assay on wing discs is not sufficiently sensitive to allow detection of small effects; PKA was found to have a modest effect on Sxl nuclear entry in the germline. Sxl is sufficiently small (38-40 kDa) to freely diffuse into the nucleus, or the protein may enter the nucleus as a complex with splicing components. This may account for the limited sex transformations caused by removal of Hh pathway components (Horabin, 2003).

Removal of several of the Hh pathway components, such as smo, gives the same weak sex transformation phenotype, even though smo has no effect on Sxl nuclear entry. Additionally, there is no correlation between a positive and a negative Hh signaling component and whether there is a resulting phenotype. Changing the dynamics of the activation state of the Hh cytoplasmic complex may perturb the normal functioning of Sxl, since Sxl appears to be in the same complex as Ci. For example, if the Hh pathway is fully activated because of a mutant condition, the relative amounts of Sxl in the cytoplasm versus nucleus at any given time, may be different from the wild-type condition. Perturbing the usual cytoplasmic-nuclear balance could compromise the various processes that Sxl protein regulates. Sxl acts both positively and negatively on its own expression through splicing and translation control and, additionally, regulates the downstream sex differentiation targets. The latter could also be responsible for the weak sex transformations seen, in view of the recent demonstration that doublesex affects the AP organizer and sex-specific growth in the genital disc (Horabin, 2003).

With the exception of Cos2, which can produce relatively substantial effects on Sxl levels in embryos as well as sex transformations in the foreleg, the effects of removal of any of the other Hh pathway components are generally not large. The strong effects of Cos2 on Sxl could be because it affects the stability of Sxl. However, Sxl depends on an autoregulatory splicing feedback loop for its maintenance making the protein susceptible to a variety of regulatory breakdowns. If Cos2 altered the nuclear entry of Sxl, for example, its removal could compromise the female-specific splicing of Sxl transcripts by reducing the amounts of nuclear Sxl. Splicing of Sxl transcripts would progressively fall into the male mode to eventually result in a loss of Sxl protein (Horabin, 2003).

Cos2 and Fu have been reported to shuttle into and out of the nucleus, and their rate of nuclear entry is not dependent on the Hh signal. That Ci and Sxl are complexed with the same Hh pathway cytoplasmic components, and share and yet have unique intracellular trafficking responses to mutations in the pathway, makes it tempting to speculate that the Hh cytoplasmic components may have had a functional origin related to intracellular trafficking that preceded the two proteins. Whether this reflects a more expanded role in regulated nuclear entry remains to be determined (Horabin, 2003).

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

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' ss generated by a mutation that causes human beta-thalassemia (Lallena, 2002).

Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding

The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (Sxl), which has two RRMs, can directly compete with the essential splicing factor U2AF65, which has three RRMs, for binding to specific Py tracts. Site-specific photocross-linking and chemical cleavage of the proteins were combined to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF65 shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF65 and Sxl can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. A model is presented to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity (Banerjee, 2003).

This systematic biochemical analysis with two proteins and three natural Py tracts revealed new information on Py-tract recognition by RRMs. Although it is possible that the nature of 5-IU cross-linking, which is efficient with only certain amino acids, may have influenced the result for a particular position, taken together, the large data set presented in this study supports a compelling trend. RRM1 is bound near the 3'-end of the Py tract, and RRM2 is bound near the 5'-end, which is consistent with all known X-ray structures of the proteins containing two RRMs. Both RRMs of Sxl and only RRM1 and RRM2 of U2AF65 together constitute the minimal Py-tract recognition domain; the RRM3 motif of U2AF65 is not cross-linked to any of the Py tracts. There are two unusual observations: (1) certain 5-IU positions are cross-linked to both RRM1 and RRM2 for all of the Py tracts tested; (2) the size of the cross-linking site for RRM2 is variable and can greatly exceed the RNA site size for other RRMs. The efficient cross-linking site of RRM1 is limited to two-four uridines at the 3'-end of the Py tract. Below, a model for Py-tract recognition is presented that explains various observations, and the biological significance of this mode of RNA recognition is discussed (Banerjee, 2003).

It is postulated that RRM1 as well as RRM2 of both proteins can bind to the Py tract in multiple registers, and RNA at the junction of RRM1 and RRM2 can form a loop of variable length, resulting in an ensemble of complexes. Although, the number of different possible RNA-protein complexes could exceed 40, assuming that each RRM contacts 4 residues in the 17-nt-long Py tract of tra, only three are dealt with in detail. The actual number of residues contacted by each RRM could be different. In complex A, both RRMs are bound to two adjacent uridine stretches, which is similar to the sharp boundary observed at the RRM junction in the X-ray structure of Sxl. In complex B, whereas the binding of RRM1 is unchanged, the binding of RRM2 is shifted by 3 nt upstream. As a consequence, residues 5-8 are looped out. In complex C, the binding of RRM1 is shifted by 1 nt upstream and of RRM2 by 2 nt, resulting in a loop of 2 nt. It should be emphasized that the size and location of the loop will vary depending on the interactions of each RRM. The complexes discussed here as well as those not shown are likely in rapid equilibrium. It is possible that various RNA-protein complexes have different binding energies. This unusual situation of multiple modes of binding likely arises because it is hard for an RRM to discriminate between adjacent uridines. Several observations led to this proposal: (1) the size of the cross-linking site is variable, which can be large for RRM2 on longer Py tracts; (2) both RRMs are cross-linked to certain residues on all of the Py tracts tested; (3) although efficient cross-linking of RRM1 is restricted to the 3'-end of the Py tract, it does not cross-link to a unique set of residues. The preference of RRM1 near the 3'-end of the Py tract could limit the number of possible complexes. (4) A lack of duplicated RRM2-RRM1 cross-linking pattern supports the possibility that in the majority of complexes a single protein molecule binds to the Py tract (Banerjee, 2003).

What does the model explain? First, it explains how certain residues can be cross-linked to both RRMs. The possibility of subpopulations of various complexes implies that a particular residue could contact either RRM1 or RRM2 in a given complex. However, the reason cross-linking of the same residue to both RRMs was observed is because the experiment reflects data from a mixture of complexes. Second, the model provides the basis for the extended site size of RRM2. Although the site size for each RRM is typically 4-7 residues for a given complex, the extended site size for RRM2 can be explained by RNA looping for certain members of the ensemble. The malleable nature of uridine-rich sequences, which are known to be largely unstructured, makes them particularly suited for adopting flexible RNA loops. Third, this model could explain previous chemical interference/protection and saturation mutagenesis data for Sxl, in which the binding site appeared larger than would have been expected for two RRMs. Fourth, in the absence of RRM3 cross-linking, the idea is favored that only two of the three RRMs of U2AF65, and both RRMs of Sxl, likely contributed to the selection of the consensus sequences. This suggestion is consistent with the interaction of RRM3 with other splicing factors such as mBBP/SF1 and SAP155. However, the possibility that RRM3, which was shown to be required for Py-tract binding, lacks appropriate amino acids for 5-IU cross-linking cannot be ruled out. Fifth, the positioning of the RRM1 of U2AF65 at the 3'-end of the Py tract would allow interaction with the small subunit (U2AF35), and thus ready recognition of the 3'-splice-site AG dinucleotide by U2AF35. Sixth, the model explains how Sxl could bind uridine tracts of variable length in the Sxl-regulated pre-mRNAs, and how U2AF65 could bind to natural Py tracts that differ widely in length. Finally, although a comparison of the cross-linking pattern and the Sxl X-ray structure indicates differences in binding, the idea is favored that the Sxl structure represents only one member of the ensemble, perhaps chosen because of the crystal contacts that favored crystallization (Banerjee, 2003).

The cross-linking pattern observed here is inconsistent with an alternative model(s) in which RRM1 and RRM2 would contact a fixed site in a single register with a sharp boundary at the junction of two RRMs. In this scenario, somehow RRM2 would contact a much larger site at the same time; this is inconsistent with the known interactions for RRMs, including the Sxl structure in which RRM2 contacts only 3 residues. Alternatively, if RRM1 and RRM2 are constrained with respect to each other upon RNA binding, the entire protein could bind at different locations. This would result in an increased site size for RRM1 and an increased number of residues cross-linked to both RRMs. The observed cross-linking pattern -- restricted cross-linking of RRM1 to the 3'-end of the Py tract and cross-linking of only 2-4 residues to both RRMs -- is incompatible with the alternative model (Banerjee, 2003).

Although recognition of a Py tract in multiple modes explains several observations, it begs the question of whether or how one member of the ensemble might convert to another. Either RRM2 could slide on the RNA with respect to RRM1 or the protein may undergo dissociation/reassociation. Also the molecular basis for the preferential cross-linking of RRM1 to the 3'-end of the Py tract is not understood; perhaps there is a signal at the 3' boundary. The exact site size for an RRM or the amount of each complex cannot be accurately determined because the observed cross-linking depends on the product of occupancy and the intrinsic cross-linking efficiency of a given binding site. It is not possible to distinguish whether RRM1 is flexible or constrained with respect to RRM2 when bound to RNA in solution. The X-ray structure shows that although two RRMs of Sxl are tethered by a flexible linker in the absence of RNA, the linker region forms a short 310-helix upon RNA binding. In addition, the RRM2 of Sxl when bound to RNA interacts with RRM1 as well as the linker region. Similar interactions are also seen for HuD. However, the energetics of these interactions for Sxl as well as the structure of the first linker region of U2AF65 when bound to RNA remain to be determined (Banerjee, 2003).

The model has important biological consequences. In general, the strength of 3'-splice sites correlates well with the length of the adjacent Py tracts, and the binding affinity for U2AF65. Two possibilities for this correlation have been envisioned. All three RRMs of U2AF65 could contact longer Py tracts, whereas only a subset of the RRMs contact shorter Py tracts. Alternatively, all three RRMs of U2AF65 could contact Py tracts, regardless of the length of the Py tract, but the number of interactions differs depending on the length of the Py tract. It was found that both RRM1 and RRM2 of U2AF65 are cross-linked to all three Py tracts, including the shortest FS Py tract of tra, and that RRM3 is not cross-linked to any of the Py tracts tested, including the longest, NSS Py tract of tra. Therefore, it is proposed that changes in the number of interactions with only RRM1 and RRM2, the number of possible complexes or both, rather than interactions with a subset of RRMs (one, two, or three RRMs), provide the most likely basis for different affinities for various-length Py tracts, and thus 3'-splice-site strength. In this scenario, longer Py tracts would provide additional registers or binding sites, thereby resulting in increased apparent binding affinity. For example, if an RNA offers a single register for binding, only one of the possible encounters with the protein will lead to productive binding; others would require continued sampling until the correct register is found. In contrast, if there are multiple correct registers, encounters with any of them will be productive, thereby increasing the chances of finding the binding site. A homopolymeric sequence like poly(U) provides a much larger set of binding sites because different registers, rather than being contiguous, extensively overlap, thereby offering a significantly large advantage in increasing the apparent binding affinity (Banerjee, 2003).

In conclusion, these studies provide insight into Py-tract recognition. These findings should be applicable to the entire family of proteins that recognize uridine-rich sequences, contain multiple RRMs, and show sequence and structural similarities with Sxl. The modified NCS cleavage protocol and the tryptophan-based domain mapping strategy described in this study provide a useful means for defining recognition of RNA, DNA, or protein sequences by any protein that has multiple recognition domains. This detailed biochemical analysis underscores the importance of independent evaluation of conclusions from structural studies (Banerjee, 2003).

Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation

MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (Sxl), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Translationally silenced msl-2 mRNPs has been purified and UNR (upstream of N-ras) has been identified as a protein recruited to the 3' UTR by Sxl. Sxl requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. These results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a 'fail-safe' dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells (Duncan, 2006).

Sequence analysis revealed that the protein specifically associated with translationally silenced msl-2 mRNA exhibited significant similarity to the previously characterized mammalian protein UNR. This was surprising for a putative translational corepressor, because mammalian UNR, a cytoplasmically localized RNA-binding protein, stimulates translation of both viral and cellular internal ribosome entry site (IRES) containing mRNAs. UNR is also a major regulator of translationally coupled mRNA turnover mediated by the c-fos mCRD RNA element (Duncan, 2006).

UNR has five cold-shock nucleic acid-binding domains, each with the unique substitution of the sequence FFH for the canonical FVH in part of the RNA-binding surface. CG7015, coding for the identified protein, also has five cold-shock domains (CSDs) with the signature FFH motif. Overall sequence identity between CG7015 and human UNR is ~45%, and this is higher within the CSDs (70%, 56%, 51%, 53%, and 66% identity for CSD-1-CSD-5, respectively). The Drosophila genome encodes no other protein with similarly high sequence identity to mammalian UNR, and it is therefore concluded that ORF CG7015 is Drosophila UNR and it is referred to as 'UNR' hereafter (Duncan, 2006).

This study has identified a novel component of the dosage-compensation regulatory machinery that has eluded genetic methods. Using an mRNP purification approach and functional analysis, the Drosophila UNR protein has been demonstrated to be recruited to msl-2 mRNA 3' UTR by SXL for translational inhibition of msl-2 mRNA specifically in female cells. These data indicate that SXL imparts a female-specific translational repressor function to UNR, and imply that this novel function of UNR is critical for negative regulation of the dosage compensation machinery to prevent toxic effects in female cells (Duncan, 2006).

Previous results implied that region 2456 of the msl-2 mRNA, 3'-UTR sequences adjacent to the Sxl-binding sites, is important for translational regulation via the 3' UTR (Grskovic, 2003). Since this region flanks the Sxl-binding sites, it was hypothesized to bind a putative corepressor that acts in conjunction with Sxl. Glutathione RNA (GRNA) chromatography in combination with sucrose-density gradient centrifugation was used to purify this factor, identifying Drosophila UNR. Functional analyses of msl-2 reporter genes and endogenous msl-2 expression in female and male cell lines demonstrate that UNR is necessary for translational repression of msl-2 mRNA by Sxl via the 3' UTR, but does not affect msl-2 mRNA translation in the absence of Sxl. Taken together, these results show that UNR is a cofactor for translational repression of MSL-2 protein synthesis, specifically in female cells. These conclusions are strongly supported by the results of Abaza, (2006), who independently isolated UNR using a different approach and could demonstrate that direct interaction of UNR with Sxl helps recruit UNR to the msl-2 3' UTR and is critical for translational inhibition of msl-2 reporter mRNAs by Sxl in vitro. This is the first time that a translational corepressor has been identified by a combined strategy of gradient and specific mRNP purification, and it is anticipated that this method will prove useful as a general strategy (Duncan, 2006).

Analysis of msl-2-ß-gal reporters further supports the recently proposed dual-mechanism model for msl-2 mRNA translational inhibition, which predicts that 3'-UTR corepressors should be required exclusively for 3'-UTR-mediated inhibition. Indeed, UNR depletion significantly affects only msl-2 reporters with wild-type 3' UTRs, and the strongest effect is on the 5'mut reporter, where all regulation must occur through the 3' UTR. In this case, the quantitative effect of UNR depletion approaches the effect of Sxl depletion. Since RNAi produces a 'knockdown' effect that likely reflects a partial-loss-of-function rather than true null phenotype, differences in RNAi efficiency and/or differences in relative concentrations of UNR and Sxl necessary for inhibition may explain why the 5'mut reporter is still slightly repressed after UNR knockdown. In any case, the in vivo analysis presented here directly supports the concept of independent regulatory contributions of 5'- and 3'-UTR Sxl complexes, and implies that UNR is a critical component for 3'-UTR-mediated inhibition (Duncan, 2006).

How does UNR recruited by Sxl to the 3' UTR interfere with translation initiation at the mRNA 5' end? Presumably the Sxl/UNR corepressor complex interacts with factors that affect small ribosomal subunit recruitment. This interaction might require direct participation of Sxl, or Sxl might serve only to recruit UNR to the 3' UTR. Similarly, UNR might directly contact factors affecting small subunit recruitment, or may do so through additional bridging factors as part of a larger 'corepressor assembly'. The biochemical approach identified factors in addition to UNR that specifically copurify with the repressed mRNP. Interestingly, UNR is the only one of the copurified proteins that displays significant corepressor activity when assayed by RNAi in Kc cells (Duncan, 2006).

Since msl-2 mRNA repression functions in the absence of a 5' m7GpppN cap structure, translational regulatory proteins like Cup or d4EHP are unlikely to be the molecular targets of repression by the 3'-UTR complex. A candidate target is Drosophila PABP, since mammalian PABP interacts with UNR, and promotes small ribosomal subunit binding to the mRNA. Although msl-2 translational inhibition does not require a poly(A) tail, PABP appears to have a critical function in initiation that is independent of the poly(A) tail, raising the possibility that UNR might nevertheless interfere with PABP-mediated recruitment of the small ribosomal subunit to msl-2 mRNA. Future studies will aim to determine the mechanism by which Sxl and UNR bound at the 3' end of msl-2 mRNA block translation initiation at the 5' end (Duncan, 2006 and references therein).

Consistent with a general role as a regulator of dosage compensation, UNR mRNA expression is ubiquitous throughout Drosophila development. Interestingly, UNR protein is expressed at similar levels in both male and female cells in culture and in flies (Abaza, 2006), but interacts with msl-2 mRNA to modulate its translation only when Sxl is present. Thus, Sxl imparts a sex-specific, mRNA-specific translational repressor function to UNR. Sex-specific modulation of UNR function by Sxl is presumably crucial for dosage compensation, which would be compromised if the abundant UNR protein in males were able to inhibit msl-2 mRNA translation (Duncan, 2006).

Sex-specific function at the cellular and organismal level can also be viewed as context-specific function at the molecular level, with Sxl acting as a context-specific modulator of UNR function. The hypothesis that UNR function is modulated by molecular context is supported by the previously determined functions of mammalian UNR, which involve different protein-interaction partners, in the context of different RNA sequence elements. Indeed, the previously reported role for mammalian UNR as a translational activator of cellular and viral IRESes made it a rather unexpected candidate for a translational corepressor. These data identify the first function for UNR in Drosophila, and demonstrate the surprising finding that UNR can also be a critical component of translational repression complexes, underscoring the importance of both protein and RNA context in modulation of UNR function in post-transcriptional control of gene expression (Duncan, 2006).

Another notable difference between UNR-mediated translational repression and the UNR functions described previously is that in the former case UNR is the recruited protein, whereas in the latter cases, high-affinity interaction of UNR with an RNA element underlies subsequent recruitment of additional proteins by UNR. This distinction has two important implications for UNR function. First, UNR's potential regulatory targets are not confined to mRNAs with high-affinity binding sites for UNR. Second, context-specific modulators such as Sxl can be expected to be key determinants of how UNR affects regulation of a particular mRNA. Detailed mechanistic and structural analysis will be essential to answer the intriguing question of how UNR can function as a translational activator in one molecular context, and a repressor in another (Duncan, 2006).

UNR depletion in Kc cells causes a significant increase in MSL-2 protein to ~20% of that in male SL-2 cells or Sxl-depleted Kc cells. Clearly, Sxl-dependent, UNR-independent inhibition mediated by the msl-2 5' UTR contributes to repression of MSL-2 protein synthesis. It was also observed that Sxl promotes reduced endogenous msl-2 mRNA levels, but UNR does not. The results warrant interpretation in the context of previous studies of transgenic flies; females expressing msl-2 transgenes lacking the 3'-UTR regulatory sequences produce detectable MSL-2 protein, but at a significantly lower level than males or females expressing transgenes with both 5'- and 3'-UTR Sxl-binding sites deleted. The lower level of MSL-2 protein made in 3'-UTR mutant females is nevertheless sufficient to promote DCC loading onto female X chromosomes. Therefore, at the organismal level, UNR, acting through the msl-2 mRNA 3' UTR, can be expected to make a significant contribution to robust repression of MSL-2 protein synthesis and prevention of deleterious activation of the X-chromosome dosage-compensation machinery in females (Duncan, 2006).

The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (Sxl) is an essential regulatory step for X-chromosome dosage compensation in Drosophila. The mammalian upstream of N-ras (UNR) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. The Drosophila homolog of mammalian UNR has been identified as a cofactor required for Sxl-mediated repression of msl-2 translation. UNR interacts with Sxl, a female-specific protein. Although UNR is present in both male and female flies, binding of Sxl to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits UNR to adjacent regulatory sequences, thereby conferring a sex-specific function to UNR. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with Sxl in translational control (Abaza, 2006).

Inhibition of msl-2 expression is essential for development of female flies; forced expression of MSL-2 causes the assembly of the DCC on both X chromosomes and lethality. The Drosophila homolog of mammalian UNR is necessary to inhibit msl-2 expression. Drosophila UNR is recruited to the 3' UTR of msl-2 mRNA by dSxl, a female-specific protein, and plays an essential role in repressing its translation. UNR associates to the 3' UTR of msl-2 mRNA in female cells and is necessary to repress msl-2 translation in vivo. Together, these data identify UNR as a regulator of dosage compensation (Abaza, 2006).

In vitro selection experiments (SELEX) indicate that human UNR binds to purine-rich regions in the mRNA, with the consensus sequences (A/G)5AAGUA/G or (A/G)8AACG and an apparent dissociation constant (Kd) of ~10 nM. Although Drosophila UNR also recognizes purine-rich sequences in the 3' UTR of msl-2 mRNA that fall within these consensus, it does so with a very poor affinity, a situation reminiscent to that of the bacterial cold-shock proteins. Binding of UNR to msl-2 mRNA requires the binding of Sxl. The observation that msl-2 RNA fragments containing mutated Sxl-binding sites, but wild-type UNR-binding sites, do not bind to either of the two proteins suggests that Sxl does not simply induce a conformational change in UNR that allows it to bind RNA. Rather, Sxl recruits UNR to bind in close proximity in the 3' UTR of msl-2 mRNA. Stable recruitment of UNR requires the interaction of UNR with both Sxl and msl-2 mRNA, as supported by the following evidence. First, UNR is not retained in the dRBD4 column unless this column is saturated with msl-2 mRNA. Second, dUNR is not retained in the mRBD column despite the presence of msl-2 mRNA. Third, no complex formation is observed in a gel mobility-shift assay when the UNR-binding sites are mutated (mut2456). Fourth, msl-2 mRNA and UNR do not interact in male flies, which lack Sxl. Nevertheless, UNR and Sxl can interact directly in vitro. Addition of EF RNA does not improve this interaction, and addition of embryo extract actually competes it. These results suggest that, although the interaction of Sxl and UNR can occur directly, the interaction with msl-2 mRNA stabilizes the complex in the competitive conditions of the extract (Abaza, 2006).

UNR protein and msl-2 mRNA do not interact in male flies despite their relative abundance. In addition, supplementing cytoplasmic embryo extracts with Sxl—a primarily nuclear protein—promotes UNR association with msl-2 mRNA, and translational repression by UNR is only observed in Sxl-containing cells. These data suggest that the interaction of UNR with msl-2 mRNA is mediated by Sxl in vivo, and imply that Sxl is the critical determinant for the formation of a repressive complex on the 3' UTR of msl-2 mRNA. In this scenario, Sxl conveys a sex-specific function to UNR. The stepwise assembly of a translation inhibitory complex on msl-2 mRNA is reminiscent of Drosophila hunchback. The 3' UTR of maternal hunchback mRNA is bound by Pumilio (Pum), and this event triggers the sequential recruitment of Nanos (Nos) and Brain tumor (Brat), which ultimately results in the translational repression of hunchback mRNA. Sequential binding of Sxl and UNR to msl-2 mRNA could result from their respective subcellular locations: While Sxl is nuclear and associates with msl-2 pre-mRNA, UNR is primarily, if not exclusively, cytoplasmic. Interestingly, UNR accumulates at the nuclear periphery, which perhaps reflects or ensures the rapid formation of repressive complexes as msl-2 and probably other mRNAs are exported to the cytoplasm. Additionally, accumulation around the nucleus could reflect the association of UNR with the endoplasmic reticulum, as reported for mammalian UNR (Abaza, 2006).

Dosage compensation is believed to function from the blastoderm stage. As expected for a protein involved in the regulation of dosage compensation, UNR is present throughout development. Curiously, although UNR mRNA is dramatically more abundant in female flies, this difference is compensated at the protein level, suggesting the existence of sex-specific mechanisms to modulate UNR expression. Indeed, the amount of UNR might need to be tightly controlled. Overexpression of mammalian UNR leads to cell death, and preliminary data suggest that substantial overexpression of UNR results in lethality of both male and female flies. Three forms of UNR mRNA can be detected in Drosophila. Several mRNAs have also been detected in mammals, consistent with the observation of three alternative polyadenylation sites of the hUNR gene and alternative splicing of the hUNR pre-mRNA. These data suggest that the different UNR mRNAs arise by alternative processing, although the significance of this observation remains to be explored (Abaza, 2006).

The role of UNR in Drosophila contrasts with the known functions of UNR in mammals. hUNR is part of a complex assembled on the coding region of c-fos mRNA that is involved in the deadenylation-dependent destabilization of this transcript. The interaction of hUNR with PABP within this complex is believed to bridge the complex to the poly(A) tail, although the mechanism by which the complex influences deadenylation is unknown. In Drosophila, the steady-state levels of msl-2 mRNA are, indeed, lower in females. However, no effect of UNR and Sxl on msl-2 mRNA stability was detected in translation assays. hUNR also binds to the IRES elements in the 5' UTRs of several transcripts and activates their translation. In the best understood example, that of Apaf-1 mRNA, hUNR induces a conformational change in the IRES that makes it accessible for binding of PTB, a positive regulator of Apaf-1 translation. Contrary to hUNR, Drosophila UNR binds to the 3' UTR of msl-2 mRNA and represses its translation. Nonetheless, the underlying effects of UNR binding may be similar if UNR acts as an RNA or RNP chaperone to facilitate an RNA conformation, or the assembly of repressive factors, that inhibit translation (Abaza, 2006).

Translation of msl-2 occurs via a cap-dependent mechanism. Cap-dependent translation initiation involves the recruitment of 43S ribosomal complexes (molecular assemblies of the 40S ribosomal subunit with a set of translation initiation factors and the initiator tRNA) to the cap structure at the 5' end of the mRNA. Translation inhibition mediated by the 3' UTR of msl-2 results from a block of 43S ribosomal recruitment. However, translational repression of msl-2 mRNA by Sxl can occur in the absence of a cap structure and a poly(A) tail. Understanding how 43S recruitment is affected by Sxl without the involvement of the cap is, indeed, intriguing. A possibility is that, as with mammalian UNR, UNR interacts with PABP. PABP could, in turn, exert a poly(A)- and cap-independent effect on translation. Certainly, the mapping of UNR domains relevant for translational control and the identification of dedicated factors that interact with UNR are likely to provide insights into this mechanism of translation regulation that is key to control dosage compensation in Drosophila (Abaza, 2006).

Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary

In the Drosophila female germline, spatially and temporally specific translation of mRNAs governs both stem cell maintenance and the differentiation of their progeny. However, the mechanisms that control and coordinate different modes of translational repression within this lineage remain incompletely understood. This study presents data showing that Mei-P26 associates with Bam, Bgcn and Sxl and nanos mRNA during early cyst development, suggesting that this protein helps to repress the translation of nanos mRNA. Together with recently published studies, these data suggest that Mei-P26 mediates both GSC self-renewal and germline differentiation through distinct modes of translational repression depending on the presence of Bam (Li, 2013).

This study presents data that Mei-P26 cooperates with Bam, Bgcn and Sxl to control the translation of nanos mRNA in the Drosophila female germline. Co-immunoprecipitation experiments indicate Mei-P26 physically associates with the differentiation factors Bam, Bgcn and Sxl and yeast 2-hybrid assays suggest the interaction between Mei-P26 and Bgcn may be direct. Disruption of mei-P26, or snf, which disrupts sxl expression in the germline, results in the upregulation of Nanos protein expression in early differentiating cysts. Both Mei-P26 and Sxl protein associate with nanos mRNA (Chau, 2012). In light of the recently published study that shows mutating Sxl binding sites within the 3′UTR of nanos mRNA leads to mis-regulation of the gene (Chau, 2012), these results suggest that Mei-P26 may be part of a Sxl, Bgcn and Bam complex that serves to promote cyst development by directly repressing the expression of Nanos. However, despite repeated attempts, direct interactions between Bam and Bgcn with nanos mRNA could not be detected. While various technical issues may prevent the detection of these specific interactions, the inability to observe direct association between Bam/Bgcn and nanos mRNA leaves open the possibility that interactions between the components of the Mei-P26, Sxl, Bam and Bgcn complex and its target mRNAs may be dynamic in nature. For instance, Bam and Bgcn may help to prepare Sxl and Mei-P26 for mRNA binding but do not themselves directly interact or only transiently interact with these targets. Further experiments will be needed to clarify the more specific molecular mechanisms that underlie Bam/Bgcn function with respect to the translational repression of nanos mRNA (Li, 2013).

Two other recent studies investigated the role of mei-P26 during germline development. Liu (2009) showed that the RNA helicase Vasa directly regulates the translation of mei-P26 mRNA through poly (U) elements within its 3' UTR. Mutations in each gene strongly enhance the phenotype of the other, resulting in the formation of cystic germline tumors. Neumuller (2008) focused on the function of Mei-P26, showing that it negatively regulates the activity of the miRNA pathway. It is now proposed that Mei-P26 functions in both GSCs and early differentiating germ cells. Within GSCs, Mei-P26 is in a complex with miRISC proteins and enhances miRNA-mediated silencing. In addition, Mei-P26 associates with Nanos protein and promotes BMP signaling within GSCs by repressing the expression of the negative regulator Brat. GSC daughters displaced away from the cap cell niche experience less BMP signaling, allowing for the expression of Bam (Li, 2013).

It is speculated that upon Bam expression, Mei-P26 switches its activity and/or its mRNA targets. This switch allows Mei-P26 to promote germline differentiation by both negatively regulating the miRNA pathway and cooperating with Bam, Bgcn and Sxl to repress the translation of specific mRNAs such as nanos. However the complex functional relationships between Mei-P26, Sxl, Bam and Bgcn remain incompletely understood. While evidence is provided that these factors can physically associate with each other under certain conditions, disruption of these genes results in two discrete phenotypes. mei-P26 and snf mutants exhibit a cystic tumorous phenotype marked by the accumulation of undifferentiated cysts that do not express A2BP1, a molecular marker present in 4-, 8- and 16-cell cysts in wild-type samples. In contrast, disruption of bam or bgcn results in the formation of single cell germ cell tumors. These phenotypic differences suggest that Bam and Bgcn carry out additional functions independent of Mei-P26 and Sxl. A more complete characterization of the regulatory networks that govern the very early steps of germline cyst differentiation will have to await a better biochemical characterization of Bam and Bgcn function (Li, 2013).

Together these data suggest that Mei-P26 has a variety of molecular functions inside and outside of the germline. It remains unclear whether Mei-P26 exhibits the same biochemical activity when complexed with different proteins or whether its function completely changes depending on context. Based on the presence of a RING domain, Mei-P26 may act as an ubiquitin ligase. However this specific enzymatic activity has not been demonstrated nor have any direct in vivo substrates been identified. In regards to the translational repression of specific mRNAs, a model is favored in which Mei-P26 exhibits the same molecular activity within GSCs and their early differentiating daughters. It is further speculated that association of Mei-P26 with different mRNA binding proteins modulates its targeting of specific mRNAs, and/or the degree to which these different targets are repressed. The expression of Bam correlates with changes in the development role of Mei-P26 but the manner in which Bam alters the composition or activity of the Mei-P26 complex remains unknown. Regardless, the findings that Bam can associate with Mei-P26 and Sxl provide further support for the hypothesis that Bam regulates the translation of specific mRNAs to promote the early steps of differentiation within the Drosophila female germline (Li, 2013).


Table of contents


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

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