Interactive Fly, Drosophila

Regulation of splicing by SXL: 1. General considerations

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. The functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions have been investigated. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains; proteins that 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. These results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. It is speculated 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. The interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain (Samuels, 1998).

Sex-lethal (Sxl) is an RNA-binding protein containing two conserved RNA binding domains (RBDs) and a glycine-rich region that functions as a regulator of alternative splicing in Drosophila sex determination. Sxl monomers interact cooperatively upon binding to target RNAs; this cooperativity depends on the glycine-rich N terminus. Band shift experiments were used to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog). Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis. Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, are suggested by the yeast two-hybrid assay. In a systematic examination of the RNA binding properties of Sxl domains, the Sxl termini as well as the RBDs influence RNA binding specificity. Selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure (Wang, 1997).

The Drosophila Sex-lethal protein is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within SXL and Transformer (TRA) pre-mRNAs. In an examination of SXL mRNA binding specificity, SXL prefers polyuridine stretches surrounded by purine residues. SXL appears to recognize and preferentially bind to a polyuridine stretch with a downstream AG sequence (Sakashita, 1994).

Sex lethal has two transcripts, early and late, with different promoters and dramatically different splicing patterns. The Sxl early transcripts are activated transiently in early embryos by a female-specific promoter and have a unique 5' exon (E1) located between late exons 1 and 2. Exon E1 is spliced to exon 4, which is common to all SXL transcripts, skipping both exons 2 and 3 (Keyes, 1992). In contrast, the late SXL transcripts derive from an essentially constitutive promoter but are spliced sex specifically. The male-specific exon, exon 3, is included by default in all male transcripts and contains in-frame nonsense codons that block SXL protein production. In the presence of SXL protein, the late transcripts skip exon 3 and splice in the female pattern. The function of the SXL early transcripts, which are present only briefly, is to generate an initial pulse of SXL protein that serves to direct splicing of the late SXL transcript into the female mode. After the initial phase, SXL female splicing and female development are maintained throughout life by SXL protein generated from the late transcripts in a positive autoregulatory loop. It is essential that both early and late SXL splicing be correctly regulated, because inappropriate levels of SXL expression in either males or females will lead to lethality arising from inappropriate X-chromosome dosage compensation. Even though the early transcripts are present briefly in early embryogenesis whereas the late transcripts are present virtually throughout life, no embryo-specific splicing factors are needed for the early splice. Neither are sex-specific factors required. Instead, the early splicing pattern is dependent on whether the 5' splice site region originates from exon E1 or exon 2 (Zhu, 1997).

As part of a cascade of genes that are regulated by sex-specific splicing, SXL controls the sex-specific splicing of Transformer (TRA) RNA and also its own RNA. SXL contains two RNA-binding domains and is known to bind TRA pre-mRNA near the alternative 3' splice site, thus blocking use of that site to give the female-specific splicing pattern. SXL not only binds SXL pre-mRNA near the alternative 3' splice site but also at distant, multiple sites surrounding the SXL alternative exon. Moreover, SXL binds cooperatively at these multiple sites. The SXL amino terminus is essential for the cooperative interaction and is also required for regulatory activity in vivo. It appears that this region of SXL protein, which resembles regions in some other RNA-binding proteins, is a domain that mediates protein-protein interactions during RNA binding and plays an important role in splicing regulation (Wang, 1994).

The RNA binding activity of SXL was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Both RNA binding domains (RBDs) are required in cis for site-specific RNA binding. Individual RBDs interact with RNA more weakly and lose the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts (Kanaar, 1995).

The mechanism for generating female Transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. In addition, a blockage mechanism is involved in Sex-lethal autoregulation. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. The splice signals at the exon are suboptimal with regard to alternate splicing (Horabin, 1993a).

Sex lethal antagonizes the general splicing factor U2AF65 to regulate splicing of TRA. Transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 were used to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on TRA splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain TRA splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of SXL pre-mRNA. These results are a direct proof that SXL, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing (Granadino, 1997).

Splicing mechanism of SXL: 2. Sxl's role in Transformer pre-mRNA splicing

Sex-specific alternative splicing of RNA from the Drosophila transformer gene involves competition between two 3' splice sites. In the absence of Sex-lethal activity (as in males), only one site functions; in the presence of Sex-lethal activity (as in females), both sites function. Information for sex-specific splice site choice is contained within the intron itself. Deletions of the splice site used in males lead to Sex-lethal-independent use of the otherwise female-specific site. The relative amounts of unspliced and spliced RNA derived from these mutant genes do not change with changes in Sex-lethal activity. Specific nucleotide changes in the non-sex-specific splice site do not affect splicing activity but eliminate Sex-lethal-induced regulation. A deletion removing material between the two splice sites does not eliminate sex-specific regulation, while a deletion of the female splice site leads to a female-specific increase in unspliced RNA. These results are consistent with a model in which female-specific factors block the function of the non-sex-specific 3' splice site (Sosnowski, 1989).

Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal, is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. This study addresses the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. Co-transfection experiments were performed in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, it was found that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing (Inoue, 1990).

The transformer gene of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage. 40 nucleotides immediately upstream of the regulated splice site are sufficient to direct sex-specific regulated splicing in transgenic animals. This entire region appears to be necessary for regulation and for efficient Sex-lethal binding. Natural splice sites containing partial homology to transformer do not show regulation. Mutations which replace the 16 nucleotides surrounding the branch point or alter single nucleotides near the splice site eliminate or reduce regulation without eliminating splicing. Mutations which reduce or eliminate regulation in vivo reduce binding to Sex-lethal in vitro, consistent with the hypothesis that these mutations bring about their effects by altering Sex-lethal binding rather than by altering binding sites for additional non-Sex-lethal factors (Sosnowski, 1994).

The Sex-lethal (Sxl) protein regulates alternative splicing of the Transformer (TRA) messenger RNA precursor by binding to the tra polypyrimidine tract during the process of sex-determination. Sxl binds tightly to a characteristic uridine-rich polypyrimidine tract at the non-sex-specific 3' splice site in one of the TRA introns, preventing the general splicing factor U2AF from binding to this site and forcing it to bind to the female specific 3' splice site. The crystal structure has now been determined at 2.6 A resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The Sxl-binding polypyrimidine tract of TRA-mRNA does not form a tertiary base-paired structure. The two RNA-binding domains have their beta-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers an insight into how a protein binds specifically to a cognate RNA without that RNA possessing any intramolecular base-pairing (Handa, 1999).

The Drosophila protein Sex-lethal (Sxl) contains two RNP consensus-type RNA-binding domains (RBDs) separated by a short linker sequence. Both domains are essential for high-affinity binding to the single-stranded polypyrimidine tract (PPT) within the regulated 3' splice site of the transformer (tra) pre-mRNA. In this paper, the effect of RNA binding to a protein fragment containing both RBDs from Sxl (Sxl-RBD1 + 2) has been characterized by heteronuclear NMR. Nearly complete (85%-90%) backbone resonance assignments have been obtained for unbound and RNA-bound states of Sxl-RBD1 + 2. A comparison of amide 1H and 15N chemical shifts between free and bound states has highlighted residues that respond to RNA binding. The beta-sheets in both RBDs (RBD1 and RBD2) form an RNA interaction surface, as has been observed in other RBDs. A significant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sxl have different or nonanalogous points of interaction with the tra PPT. R142 (in RBD2) exhibits the largest chemical shift change upon RNA binding. The role of R142 in RNA binding was tested by measuring the Kd of a mutant of Sxl-RBD1 + 2 in which R142 was replaced by alanine. This mutant lost the ability to bind RNA, showing a correlation with the chemical shift difference data. The RNA-binding affinities of two other mutants, F146A and T138I, were also shown to correlate with the NMR observations (Lee, 1997).

Table of contents

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

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