An N-terminal truncation uncouples the sex-transforming and dosage compensation functions of sex-lethal.

Sex-lethal (Sxl) encodes an RNA recognition motif (RRM) class RNA binding protein that serves as the developmental switch for sex determination in Drosophila melanogaster (6). Sxl is expressed only in females, where it controls sexual differentiation and dosage compensation by posttranscriptional regulatory mechanisms that affect pre-mRNA splicing and mRNA translation. Misregulation of Sxl results in sex-specific lethality and sex transformations (see reference 15 and references therein). Female sexual identity is maintained by an autoregulatory feedback loop in which Sxl proteins promote their own synthesis by directing the female-specific splicing of Sxl pre-mRNAs (Fig. ​(Fig.1A;1A; references 5, 6, and 14). Functional female Sxl mRNA is generated by joining exon 2 to exon 4, skipping the third (male-specific) exon, which contains in-frame translation stop codons. Male identity is maintained by the default splicing machinery, which incorporates the third exon into the mature mRNAs, ensuring that no Sxl protein is produced. Sxl-dependent posttranscriptional regulation also controls the gene cascades that direct the different aspects of female or male development (Fig. ​(Fig.1A).1A). Sxl protein promotes female differentiation by directing the female-specific splicing of transformer (tra) pre-mRNAs (25, 45, 46). In the absence of Sxl protein, the default splicing of tra results in mRNAs that do not encode functional protein. Sxl also regulates dosage compensation, which is responsible for equalizing the expression of X-linked genes in the two sexes. One component of the dosage compensation system is the hyperactivation of X-chromosome gene expression in males by the male specific lethal (msl) genes (1, 7, 32, 33). Sxl proteins prevent hyperactivation in females by blocking both the splicing and translation of transcripts from one of these genes, msl-2 (3, 18, 28, 51). A second component of the dosage compensation system is msl independent (8, 19, 20) and is thought to function in females to reduce X-chromosome gene expression. Kelley et al. (28) recently suggested that Sxl itself mediates this dosage compensation by repressing the translation of mRNAs expressed from X-linked genes. FIG. 1 Regulatory activities of Sxl. (A) Models for Sxl splicing, tra splicing, and msl-2 translational control in Drosophila males (top) and females (below). The default splicing of both Sxl and tra occurs in males and results in transcripts with in-frame stop ... The posttranscriptional regulatory activities of the Sxl gene depend on direct interactions between Sxl proteins and target RNAs. RNA binding activity is provided by Sxl’s two RRM domains, R1 and R2 (26, 37, 43, 49). The two RRM domains recognize poly(U) runs of seven or more nucleotides, and all of the known Sxl regulatory targets have one or more of these arrays. In the case of tra, in vivo and in vitro studies indicate that Sxl protein directs female-specific splicing by binding to a poly(U) run in the polypyrimidine tract of the default 3′ splice site (Fig. ​(Fig.1A)1A) (25, 45, 46). It has been proposed that this prevents the generic splicing factor U2AF from binding to the default polypyrimidine tract and forces the assembly of a U2AF-U2 snRNP splicing complex on the weaker, female-specific 3′ splice site downstream (22, 48). While a direct competition for overlapping binding sites accounts for what is known about tra splicing, the RNA binding activity of Sxl is not sufficient to explain either Sxl autoregulation or the repression of msl-2 translation. The key targets for Sxl autoregulation are located in the introns upstream and downstream of the male exon at distances of 200 or more nucleotides from the regulated 3′ and 5′ splice sites (24). Hence, instead of a direct blockage mechanism, Sxl must indirectly prevent the assembly of productive splicing complexes at the male exon. One possibility is that homotypic interactions between Sxl proteins sequester the male exon from the splicing machinery (24, 35). Consistent with this possibility, Sxl proteins interact in vitro, and these interactions stabilize Sxl complexes on RNA (26, 36, 43, 49). These Sxl-Sxl interactions are mediated by the two RRM domains (37, 42, 50). A second model postulates that Sxl interacts with and inactivates components of the splicing machinery assembled at the male exon splice sites (e.g., U1 and U2 snRNPs [16, 40]). Consistent with this model, Sxl proteins in vivo are found in large complexes which contain both U1 and U2 snRNPs and Sxl pre-mRNAs (16, 43). In addition, mutations in the sans-fille (snf) gene, encoding the fly homologue of two mammalian snRNP proteins, U1A and U2B", disrupt autoregulation and exacerbate the female-lethal effects of Sxl mutations (38, 40). This synergism may be attributed to interactions between these two proteins; Snf-Sxl complexes can be detected in vivo and in vitro, and this interaction is mediated by the first Sxl RRM domain (16, 43). Finally, efficient translational repression of msl-2 mRNA requires Sxl protein binding sites in both the 5′ and 3′ untranslated regions (UTRs) (4, 18, 29). In a manner analogous to autoregulation, interactions between Sxl proteins upstream and downstream of the open reading frame could sequester the msl-2 mRNA from the translational machinery. Alternatively, Sxl might interact with and poison this machinery. While the two Sxl RRM domains have been implicated in both RNA binding and protein-protein interactions, much less is known about the functions of the N- and C-terminal domains. Though there are no known mutations in the N-terminal domain, there are some indications that it may be important for the regulatory functions of the Sxl protein. A truncated Sxl protein was detected in the heads of adult D. melanogaster males (11). This smaller isoform appears to result from translation initiation at an AUG codon in exon 4, downstream of the male exon (exon 3), and gives a protein lacking the first 40 amino acids (aa). A slightly larger male-specific protein is also detected in the related drosophilid, D. virilis (12). Although the D. melanogaster and D. virilis male proteins contain both RRM domains and appear to bind appropriate target RNAs, they do not have detectable feminizing activities. It was initially thought that the concentration of the truncated proteins might be too low to induce feminization. This explanation was called into question by Wang and Bell (49), who found that a truncated Sxl protein (SxlN1), similar to one observed in D. melanogaster male heads, was impaired in Sxl autoregulation when transiently expressed in tissue culture cells. To learn more about the regulatory functions of the different Sxl protein domains, we have compared the biological activities of the full-length Sxl protein (Sx.FL) and Sx-N in vivo. We were able to uncouple the splicing and translational regulatory activities of Sxl protein. Sx-N is impaired in autoregulatory function, and contrary to the expectations of the U2AF blockage model, the N terminus plays an essential role in the regulation of tra splicing. However, these amino acids are not required to regulate msl-dependent dosage compensation. Finally, we provide evidence that Sxl is controlled by both positive and negative autoregulation.