Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal - PubMed (original) (raw)
Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal
B Granadino et al. Proc Natl Acad Sci U S A. 1997.
Abstract
The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 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.
Figures
Figure 1
(a) Schematic representation of the exons and introns of tra and Sxl pre-mRNAs that are relevant for the regulated patterns of splicing controlled by the protein SXL. In the absence of SXL (male flies) the non-sex-specific (nss) 3′ splice site of tra is used, and the male-specific (ms) exon of Sxl is included. The presence of SXL in female flies induces the partial use of the female-specific (fs) site in tra and skipping of the ms exon in Sxl. (b) Representation of the structural domains of Sxl, U2AF65, USx, and RS–Sx proteins. Sxl is composed of two RNP consensus motifs (RNPa and RNPb) and an NH2-terminal glycine/asparagine-rich (GN) region. U2AF65 is composed of three RNP consensus motifs that form its RNA binding domain and an NH2-terminal region—the effector domain—composed of two subdomains: an arginine/serine-rich (RS) motif and a region involved in protein–protein interactions (PI).
Figure 4
Effect of USx and RS–Sx transgenes on splicing of the Sxl–lacZ reporter gene. Transgenic flies with the indicated genotypes were maintained at 37°C for 90 min to allow expression of the transgenes, then RNA was isolated, and RT-PCR was performed to quantify the ratio between the alternatively spliced products. The positions of the RT-PCR products corresponding to the male- and female-specific spliced transcripts are indicated.
Figure 2
Effect of USx and RS–Sx transgenes on the splicing of tra pre-mRNA. Transgenic flies with the indicated genotypes were maintained at 37°C for 90 min to allow expression of the transgenes, and RNA was isolated after 48 h at 25°C. RT-PCR was then performed to quantify the ratio between the alternatively spliced products. The positions of the RT-PCR products corresponding to the non-sex-specific and female-specific spliced transcripts are indicated. SxlcF1 refers to a female Sxl cDNA transgene (13).
Figure 3
(a) Effect of the _Sxl-Δ_GN transgene on tra pre-mRNA splicing in vivo. The analysis was performed as in Fig. 2. (b) Binding of recombinant SXL and SXL-ΔGN to tra non-sex-specific 3′ splice site. Mobility-shift assays with the indicated purified recombinant SXL derivatives at a protein concentration of 5 × 10−8 M were performed. The position corresponding to the migration of unbound RNA is indicated. (c) Regulation of tra alternative 3′ splice site choice by SXL and SXL-ΔGN-in vitro. In vitro splicing reactions in the presence of the indicated purified recombinant proteins was performed and analyzed by primer extension. The products of each primer-extension reaction, corresponding to each of the spliced RNAs, are indicated.
Figure 5
(A) Effect of the USx and RS–Sx transgenes on male viability. The transgenic flies were raised at 29°C throughout development and during the second larval instar they were heat-shocked for 90 min at 37°C. Viability of males heterozygous for the transgenes and carrying a duplication of Sxl+ is shown. The crosses were USx/USx or RS–Sx/RS–Sx females and y cm Sxl fP7BO; Dp(1;3)sn 13a1 , Sxl+/TM3, Sb Ser males. In both crosses, the control was females carrying the duplication of Sxl. The number of control flies was 170 and 276 for USx and RS–Sx transgenes, respectively. Viability of males homozygous for the transgenes and carrying a deficiency of Sxl+ is shown. The crosses were cm Sxl f1 ct 6/+; USx/TM3, Sb females and B S Y; USx/USx males, or cm Sxl f1 ct 6/+; RS–Sx/TM3, Sb females and B S Y; RS–Sx/RS–Sx males. In both crosses, the control was females homozygous for the transgene and with two Sxl+ copies. The number of control flies was 122 for both USx and RS–Sx transgenes. In A, males _Sxl_− stand for males carrying the null Sxl f1 mutation. (B) Schematic representation of the effect of USx and RS–Sx transgenes on the viability of males. For the endogenous Sxl primary transcript, only the second and fourth exons (open boxes) and the male-specific third exon (solid box) were drawn. For explanations see text.
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