RNA binding protein sex-lethal (Sxl) and control of Drosophila sex determination and dosage compensation.

In the past two decades, scientists have elucidated the molecular mechanisms behind Drosophila sex determination and dosage compensation. These two processes are controlled essentially by two different sets of genes, which have in common a master regulatory gene, Sex-lethal (Sxl). Sxl encodes one of the best-characterized members of the family of RNA binding proteins. The analysis of different mechanisms involved in the regulation of the three identified Sxl target genes (Sex-lethal itself, transformer, and male specific lethal-2) has contributed to a better understanding of translation repression, as well as constitutive and alternative splicing. Studies using the Drosophila system have identified the features of the protein that contribute to its target specificity and regulatory functions. In this article, we review the existing data concerning Sxl protein, its biological functions, and the regulation of its target genes.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2)d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

Sexual back talk with evolutionary implications: stimulation of the Drosophila sex-determination gene sex-lethal by its target transformer

We describe a surprising new regulatory relationship between two key genes of the Drosophila sex-determination gene hierarchy, Sex-lethal (Sxl) and transformer (tra). A positive autoregulatory feedback loop for Sxl was known to maintain somatic cell female identity by producing SXL-F protein to continually instruct the target gene transformer (tra) to make its feminizing product, TRA-F. We discovered the reciprocal regulatory effect by studying genetically sensitized females: TRA-F from either maternal or zygotic tra expression stimulates Sxl-positive autoregulation. We found female-specific tra mRNA in eggs as predicted by this tra maternal effect, but not predicted by the prevailing view that tra has no germline function. TRA-F stimulation of Sxl seems to be direct at some point, since Sxl harbors highly conserved predicted TRA-F binding sites. Nevertheless, TRA-F stimulation of Sxl autoregulation in the gonadal soma also appears to have a cell-nonautonomous aspect, unprecedented for somatic Sxl regulation. This tra-Sxl retrograde regulatory circuit has evolutionary implications. In some Diptera, tra occupies Sxl's position as the gene that epigenetically maintains female identity through direct positive feedback on pre-mRNA splicing. The tra-mediated Sxl feedback in Drosophila may be a vestige of regulatory redundancy that facilitated the evolutionary transition from tra to Sxl as the master sex switch.     

Sex-lethal in neurons controls female body growth in Drosophila

Sexual size dimorphism (SSD), a sex difference in body size, is widespread throughout the animal kingdom, raising the question of how sex influences existing growth regulatory pathways to bring about SSD. In insects, somatic sexual differentiation has long been considered to be controlled strictly cell-autonomously. Here, we discuss our surprising finding that in Drosophila larvae, the sex determination gene Sex-lethal (Sxl) functions in neurons to non-autonomously specify SSD. We found that Sxl is required in specific neuronal subsets to upregulate female body growth, including in the neurosecretory insulin producing cells, even though insulin-like peptides themselves appear not to be involved. SSD regulation by neuronal Sxl is also independent of its known splicing targets, transformer and msl-2, suggesting that it involves a new molecular mechanism. Interestingly, SSD control by neuronal Sxl is selective for larval, not imaginal tissue types, and operates in addition to cell-autonomous effects of Sxl and Tra, which are present in both larval and imaginal tissues. Overall, our findings add to a small but growing number of studies reporting non-autonomous, likely hormonal, control of sex differences in Drosophila, and suggest that the principles of sexual differentiation in insects and mammals may be more similar than previously thought.     

Males and females: Creating differences while maintaining the similarities

During metazoan development, a small number of signaling pathways are iteratively used to orchestrate diverse processes such as cell division, cell fate specification and survival. Temporal and spatial regulation of these pathways underlies the final cellular makeup, size and shape of organs. In Drosophila melanogaster, the master switch gene Sex-lethal (Sxl) orchestrates all aspects of female development and behavior by modulating gene expression. Many of the sex-specific differences in gene expression and morphology are controlled through a gene activity cascade that involves Sxl→tra→dsx-fru. However, various aspects of somatic sexual development appear to be independent of this cascade. Consistent with this idea, Sxl protein, on its own, was recently implicated in the regulation of both Hh and Notch signaling to shape some of the sexually dimorphic traits. Paradoxically, however, Sxl activity is essential in every female cell to prevent the activation of the male-specific dosage compensation system and thus to ensure the proper level of X-linked gene expression. This raises a key question as to how the sex-specific effects of Sxl on major signaling pathways are prevented in monomorphic tissues during female development. We have elucidated a novel mechanism where Hrp48, an abundant essential hnRNP functions to restrict Sxl expression in monomrphic tissues and thus allow for proper development. Our findings bring into focus the critical role played by general homeostatic factors in specification of diverse cell fates and morphogenesis.     

Receptor tyrosine kinases mediate cell-cell interactions during Drosophila development

In vertebrates, receptor tyrosine kinases (RTKs) have been identified as growth factor receptors and proto-oncogenes. Many of these RTKs appear to play a key role in the regulation of cell growth. Recent analyses of several Drosophila genes encoding putative RTKs indicate that this class of proteins also serves an important role in cell fate decisions which depend on cellular interactions during development. The sevenless RTK mediates the position-dependent specification of a particular photoreceptor cell type (R7) in the eye. The local specification of R7 cells requires a functional tyrosine kinase domain of the sevenless protein but does not depend on the spatially restricted expression of the sevenless gene. The Drosophila EGF receptor homolog serves multiple functions during development, some of which are clearly unrelated to regulation of cell growth. Finally, the torso gene encodes an RTK required for the specification of the terminal regions of the Drosophila larva. A number of other genes have been genetically identified that appear to function in the same developmental processes upstream or downstream of these three RTKs. These loci are excellent candidates for genes encoding other components of the signalling pathways such as ligands or substrates of the RTKs.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.