Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2

Abstract

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5' splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5' splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5' splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5' splice site and the female-specific doublesex (dsx) 3' splice site, suggesting that the mechanisms of 5' splice site activation and 3' splice site activation may be similar.

FIG. 1

Patterns of fru and dsx sex-specific splicing. Schematic drawings of fruitless (fru) and doublesex (dsx) splicing patterns are shown. Only part of the fru gene is depicted. Exons (boxes), introns (thin horizontal lines), and splices (thick lines) are indicated. Female-specific splicing patterns are indicated above and male-specific splicing patterns are indicated below each gene. Male- (M) and female (F)-specific exon segments and common exons (C) are indicated. The tra/tra-2 repeat elements are indicated by thick dashes, and distances of the tra/tra-2 repeat elements to the sex-specific splice sites are given. Female-specific (AUG^F) and male-specific (AUG^M) translational start codons as well as female-specific (AA^F) and male-specific (AA^M) polyadenylation sites are shown.

FIG. 2

Regulation of fru sex-specific splicing in transfected Schneider cells. (A) The fru minigene construct fruM+Fwt was transfected together with various combinations of cDNA expression constructs encoding tra, tra-2, rbp1, and B52 as indicated (28, 53) into Schneider cells, and fru splicing was analyzed by RNase protection assays. The presence (+) or absence (−) of construct fruM+Fwt and of the tra, tra-2, rbp1, and B52 expression constructs in the transfection experiments is indicated above each lane. Splicing products are identified to the right of the autoradiograph. Lane M contains pSK+ DNA cut with HpaII and used as a nucleotide length marker. The positions of size markers (in nucleotides) are shown to the left of the autoradiographs in panels A and B. (B) The amount of cotransfected tra and tra-2 expression constructs was titrated down to 0.3% (0.1 ng) of the amount transfected in panel A. (C) Depiction of construct fruM+Fwt. Exons are represented by boxes, and the intron is represented by a thin horizontal line. The positions of the male-specific and female-specific 5′ SSs are indicated, and male-specific (M) and female-specific (F) exon segments are identified. The three dashes upstream of the female-specific 5′ SS represent the tra/tra-2 repeat elements. The antisense RNase protection probes are indicated directly below the map by horizontal bars. RNase protection products (prod.) derived from male-specific and female-specific fru splicing are given at the bottom of the panel, and the expected product lengths are given in parentheses. The distance between the male-specific and female-specific fru 5′ SSs is 1,590 nt. The intron is 1,300 nt long. Note that the RNase protection probes and the protection products are not drawn to scale. Protection product MF can be obtained as a consequence of female splicing or use of cryptic 5′ splice sites or from unspliced pre-mRNA.

FIG. 3

cis-acting elements required for regulation of fru splicing by tra/tra-2. (A) Constructs carrying mutations within the tra/tra-2 repeat elements (construct fruM+FREmut) or a deletion of sequences between the male-specific fru 5′ SS and the tra/tra-2 repeat sequences (construct fruM+FΔB-M) were transfected in the absence (−) or presence (+) of cotransfected tra/tra-2 as indicated above the RNase protection assay autoradiograph. RNase protection products are indicated to the right of the autoradiograph and are as described in the legend to Fig. 2C. An additional RNase protection product (RE mut.) is derived from female-specific splicing of construct fruM+REmut. An incompletely cleaved protection product derived from female-specific splicing of construct fruF+MRE mut is indicated by an asterisk. The positions of size markers (in nucleotides) are shown to the left of the autoradiograph. Lane M contains pSK+ DNA cut with HpaII. (B) Illustration of constructs fruM+FREmut and fruM+FΔB-M. fruM+FREmut contains mutated tra/tra-2 repeat elements indicated by asterisks. In fruM+FΔB-M, a 1-kb BbsI-MscI fragment indicated by a broken-line box was deleted. The probe used in these experiments is as described in the legend to Fig. 2.

FIG. 4

Testing the activation and the blockage model of fru regulation. (A) Construct fruFwt containing the female-specific fru 5′ SS and the tra/tra-2 repeat elements was transfected in the absence (−) or presence (+) of cotransfected tra/tra-2 as indicated above the RNase protection assay autoradiograph. Lanes 3 and 4 show a shorter exposure of lanes 1 and 2, respectively. Lane M contains pSK+ DNA cut with HpaII. The positions of size markers (in nucleotides) are shown to the left of the autoradiographs in panels A and B. (B) Construct fruM+REds containing the male-specific fru 5′ SS and the tra/tra-2 repeat elements was transfected in the absence (−) or presence (+) of cotransfected tra/tra-2 as indicated above the RNase protection assay autoradiograph. RNase protection products derived from spliced and unspliced pre-mRNAs of constructs fruFwt and fruM+REds are indicated to the right of the autoradiograph. (C) Illustration of constructs fruFwt and fruM+REds. fruFwt contains the female-specific fru 5′ SS, and fruM+REds contains the male-specific fru 5′ SS. The tra/tra-2 repeat elements were included at the correct distance upstream of the female-specific 5′ SS and downstream of the male-specific 5′ SS, respectively. The deletion of the female-specific fru 5′ SS in construct in fruM+REds is indicated by parentheses. The probes used in the RNase protection experiments are represented by horizontal bars below each construct. RNase protection products corresponding to spliced and unspliced pre-mRNAs are shown below each construct.

FIG. 5

Activation of a heterologous 5′ SS by tra/tra-2. (A) Construct fruM+REwt containing wt tra/tra-2 repeat elements upstream of the male-specific fru 5′ SS and construct fruM+REmut containing mutant tra/tra-2 repeat elements upstream of the male-specific fru 5′ SS were transfected in the absence (−) or presence (+) of cotransfected tra/tra-2 as indicated above the RNase protection assay autoradiograph. RNase protection products derived from spliced and unspliced pre-mRNAs are indicated to the right of the autoradiograph and are as described in the legend to Fig. 5B. Lane M contains pSK+ DNA cut with HpaII. The positions of size markers (in nucleotides) are shown to the left of the autoradiograph. (B) Illustration of constructs fruM+REwt and fruM+REmut. The wt fru repeat region is indicated by three dashes, and the mutated fru repeat region is indicated by three asterisks. The repeat mutations in construct fruM+REmut are as in construct fruM+FREmut. For each construct, a specific RNase protection probe hybridizing across the splice sites was generated and is indicated by a horizontal bar below the constructs. RNase protection products corresponding to spliced and unspliced pre-mRNAs are shown below the constructs.

FIG. 6

The fru and dsx repeat regions are functionally interchangeable. (A) Construct dsxF+dsxRE containing the dsx repeat region downstream of the female-specific dsx 3′ SS and construct dsxF+fruRE containing the fru repeat region downstream of the female-specific dsx 3′ SS were transfected in the absence (−) or presence (+) of cotransfected tra/tra-2 as indicated above the RNase protection assay autoradiograph. RNase protection products derived from spliced and unspliced pre-mRNAs are indicated to the right of the autoradiograph and are as shown in panel B. The slightly lower mobility of the protection product F derived from construct dsxF+fruRE (lanes 3 and 4) compared to that of product F derived from construct dsxF+dsxRE (lanes 1 and 2) is due to a short stretch of polylinker present in construct dsxF+fruRE and in the probe, but not in construct dsxF+dsxRE. Lane M contains pSK+ DNA cut with HpaII. The positions of size markers (in nucleotides) are indicated to the left of the autoradiograph. (B) Illustration of constructs dsxF+dsxRE and dsxF+fruRE. The fru repeat region is indicated by three dashes, and the dsx repeat region is indicated by six dashes. The common (C) and the female (F) dsx exon are indicated (compare diagram with that in Fig. 1). The probe used in the RNase protection experiments is represented by a horizontal bar below the constructs. RNase protection products corresponding to spliced and unspliced pre-mRNAs are shown at the bottom of the panel.