Biochemical and functional analysis of Drosophila-sciara chimeric sex-lethal proteins

Abstract

Background: The Drosophila SXL protein controls sex determination and dosage compensation. It is a sex-specific factor controlling splicing of its own Sxl pre-mRNA (auto-regulation), tra pre-mRNA (sex determination) and msl-2 pre-mRNA plus translation of msl-2 mRNA (dosage compensation). Outside the drosophilids, the same SXL protein has been found in both sexes so that, in the non-drosophilids, SXL does not appear to play the key discriminating role in sex determination and dosage compensation that it plays in Drosophila. Comparison of SXL proteins revealed that its spatial organisation is conserved, with the RNA-binding domains being highly conserved, whereas the N- and C-terminal domains showing significant variation. This manuscript focuses on the evolution of the SXL protein itself and not on regulation of its expression.

Methodology: Drosophila-Sciara chimeric SXL proteins were produced. Sciara SXL represents the non-sex-specific function of ancient SXL in the non-drosophilids from which presumably Drosophila SXL evolved. Two questions were addressed. Did the Drosophila SXL protein have affected their functions when their N- and C-terminal domains were replaced by the corresponding ones of Sciara? Did the Sciara SXL protein acquire Drosophila sex-specific functions when the Drosophila N- and C-terminal domains replaced those of Sciara? The chimeric SXL proteins were analysed in vitro to study their binding affinity and cooperative properties, and in vivo to analyse their effect on sex determination and dosage compensation by producing Drosophila flies that were transgenic for the chimeric SXL proteins.

Conclusions: The sex-specific properties of extant Drosophila SXL protein depend on its global structure rather than on a specific domain. This implies that the modifications, mainly in the N- and C-terminal domains, that occurred in the SXL protein during its evolution within the drosophilid lineage represent co-evolutionary changes that determine the appropriate folding of SXL to carry out its sex-specific functions.

Figure 1. Scheme showing the sex-specific functions of the Drosophila SXL protein.

Normal and dashed lines indicate active and inactive interactions, respectively. The crossed boxes for SXL, TRA and MSL-2 proteins designate lack of these proteins. After blastoderm stage, Sxl begins to function in both sexes, and production of the Sxl transcripts persist throughout the remainder of development and adult life. The male-specific transcripts are similar to their female-specific counterparts, except for the presence of an additional exon (exon 3), which contains translational stop codons. Consequently, male transcripts give rise to presumably inactive truncated proteins. In females, this exon 3 is spliced out and functional SXL protein is produced , . The gene tra is transcribed in both sexes but its pre-mRNA follows an alternative splicing. In males, exon 2 introduces a translational stop codon, leading to the production of a truncated, presumably non-functional TRA protein. In females, however, approximately half of the tra pre-mRNA is spliced differently due to the intervention of the SXL protein, so that the RNA fragment on exon 2 containing the translation stop codon is not incorporated into the mature mRNA encoding the whole, functional TRA protein –. The gene msl-2 is transcribed in both sexes but its pre-mRNA follows an alternative splicing. In females, the SXL protein prevents the splicing of an exon at the 5′ UTR, which introduces SXL-binding sequences –. Consequently, SXL binds to these sequences and to those located at the 3′UTR inhibiting the translation of the msl2- mRNA and then MSL2 protein is not synthesised , . In males, however, the exon at the 5′ UTR is spliced out and MSL2 protein is produced.

Figure 2. Biochemical characterization of SXL proteins.

Quantitative analysis of the EMSA’s (an example is shown in Figure S1) for studying the properties of the SXL proteins to bind to RNA ligands carrying either single (grey) or double (black) poly(U) sequences. The fraction of bound RNA was quantified and plotted as a function of SXL protein concentration. Solid lines correspond to the best fit of Hill eqn.1 to the binding data obtained from titration of RNAs, with the best-fit parameters written in Table 1. “F” is defined by Eqn. 1, which is described in Materials and Methods.

Figure 3. Effect of the SXL proteins on the sex-specific splicing of endogenous Sxl (A) and tra pre-mRNAS (B) in males carrying a null allele of Sxl.

The conditions and primers for the RT-PCRs are described in Materials and Methods. The constitution of the SXL proteins is described in the Tables; and “melano m” and “melano f” stand for Drosophila wild type male and female, respectively. The genotypes of the males were: Sxm stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/+; Sxm/Tub-Gal80^ts; Sxs stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/+; Sxs/Tub-Gal80^ts; Sx17 stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/+; Sx17/Tub-Gal80^ts; Sx64 stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/Sx64; Tub-Gal80^ts/+; Sx35 stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/Sx35; Tub-Gal80^ts/+; and Sx28 stands for males ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/Sx28; Tub-Gal80^ts/+. These males were produced by crossing females ywSxl^f1ct⁶/Y; arm-Gal4,w⁺/CyO,Cy; Tub-Gal80^ts/MKRS,Sb with males yw/Y; Sxm/MKRS,Sb; males yw/Y; Sxs/MKRS,Sb; males yw/Y; Sx17/MKRS,Sb; males yw/Y; Sx64/CyO,Cy; males yw/Y; Sx35/CyO,Cy; and males yw/Y; Sx28/CyO,Cy.