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Review
. 2003 Sep;67(3):343-59, table of contents.
doi: 10.1128/MMBR.67.3.343-359.2003.

RNA binding protein sex-lethal (Sxl) and control of Drosophila sex determination and dosage compensation

Luiz O F Penalva  1 , Lucas Sánchez
Affiliations
Review

RNA binding protein sex-lethal (Sxl) and control of Drosophila sex determination and dosage compensation

Luiz O F Penalva et al. Microbiol Mol Biol Rev. 2003 Sep.

Abstract

In the past two decades, scientists have elucidated the molecular mechanisms behind Drosophila sex determination and dosage compensation. These two processes are controlled essentially by two different sets of genes, which have in common a master regulatory gene, Sex-lethal (Sxl). Sxl encodes one of the best-characterized members of the family of RNA binding proteins. The analysis of different mechanisms involved in the regulation of the three identified Sxl target genes (Sex-lethal itself, transformer, and male specific lethal-2) has contributed to a better understanding of translation repression, as well as constitutive and alternative splicing. Studies using the Drosophila system have identified the features of the protein that contribute to its target specificity and regulatory functions. In this article, we review the existing data concerning Sxl protein, its biological functions, and the regulation of its target genes.

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Figures

FIG. 1.
FIG. 1.
The X:A signal and the control of sex determination, sexual behavior, and dosage compensation. The X:A signal targets the Sxl gene, controlling its expression. Autoregulation of Sxl is established only in embryos whose chromosomal constitution is 2X;2A (two X chromosomes; two sets of autosomes), but not in X(Y);2A (one X chromosome; two sets of autosomes) embryos. The autoregulatory feedback loop regulates Sxl expression throughout development and adult life. Sxl controls the expression of the tra and msl-2 genes, whose products are required for control of somatic sex determination/sexual behavior and dosage compensation, respectively. The dotted lines indicate that the expression of the gene is “off,” and the solid lines indicate that the expression of the gene is “on”.
FIG. 2.
FIG. 2.
The somatic sex determination/sexual behavior cascade. A hierarchical interaction exists among the genes that form the backbone of the somatic sex determination/sexual behavior cascade. The product of a gene controls the sex-specific splicing of the pre-mRNA from the downstream gene in the genetic cascade. (A) The ratio between X chromosomes and autosomes (X:A signal) initiates the cascade by activating the expression of the Sxl gene. This activation occurs only in embryos that have the chromosomal constitution 2X;2A. Sxl regulates the splicing of its own pre-mRNA, a positive-feedback loop (8). The products of the fl(2)d, vir, snf, and spf45 genes are also necessary for this splicing regulation (42, 51, 52, 68, 101). The downstream target of Sxl is the tra gene; splicing control by Sxl allows the production of functional protein product (11). Tra forms a heterodimer with the Transformer-2 (Tra-2) protein (2) that modulates the splicing of two genes: double sex (dsx) (18, 56, 58) and fruitless (fru) (49, 60, 93). The generated sex-specific products control the expression of target genes necessary for female sex differentiation and behavior. (B) In X(Y);2A embryos, no Sxl protein is produced. As a consequence, tra RNA follows a different splicing pattern and no functional product is generated. fru and dsx produce male-specific transcripts. FruM and DsxM control the expression of target genes necessary for male sex differentiation and behavior. hermaphrodite (her) (91, 92) and intersex (20) are also required for proper sex differentiation. The dissatisfaction (dsf) gene is implicated in both male and female sexual behavior (31, 32). CNS, central nervous system.
FIG. 3.
FIG. 3.
Sxl and its target genes. Sxl controls the expression of tra (A) and msl-2 (B). Boxes represent exons, and horizontal lines represent introns. (A) A non-sex-specific transcript is generated when the proximal 3′ splice site is used. Use of this splice site introduces a stop codon in the open reading frame, leading to the production of a truncated and nonfunctional peptide. In females, about half of the tra pre-mRNA is spliced in a different manner due to the intervention of Sxl that competes with U2AF for binding to the poly(Y) tract associated with the proximal 3′ splice site: U2AF is then diverted to the distal poly(Y) tract, thus promoting the usage of the female-specific 3′ splice site. (B) Inhibition of msl-2 expression in females occurs in two steps. First, Sxl prevents the splicing of the first intron by competing with U2AF and Rox8 for binding to two poly(U) sequences located at the 5′ and 3′ ends of this intron. Later, binding of Sxl to these poly(U) sequences and to poly(U) stretches located at 3′ UTR will inhibit translation.
FIG. 4.
FIG. 4.
Regulation of Sxl expression. The primary genetic X:A signal acts on the early Sxl promoter and controls Sxl expression at the transcription level (67, 124). Due to a twofold difference in the number of X chromosomes (autosomes are the same in both sexes), Sxl transcription is either initiated in females but not in males or initiated in both sexes but much more efficiently in females than in males. As a result, early Sxl protein is abundantly produced in females whereas it remains undetectable in males. After the blastoderm stage, the late Sxl promoter starts functioning in both sexes, and production of the late Sxl transcripts persists throughout the remainder of the fly's life. In females, the abundant early Sxl protein imposes the female-specific splicing pathway on the late Sxl RNA, leading to the production of late Sxl protein, and the feedback loop is established. In male individuals in which no (or insufficient) early Sxl protein is produced, a different splicing pattern takes place and a truncated and nonfunctional version of Sxl is generated.
FIG. 5.
FIG. 5.
Molecular organization of the Sxl gene. The Sxl gene produces two separate sets of transcripts, linked to the function of its two promoters, the so-called early (PE) and late (PL) promoters. Alternative splicing and different polyadenylation sites give rise to several different transcripts. Boxes represent exons, and horizontal lines represent introns. The embryo-specific exon (E1) is represented in green (light green for the noncoding region, and dark green for the coding region). The male-specific exon is represented in red. Other exons are represented in blue (light blue for the noncoding region, and dark blue for the coding region).
FIG. 6.
FIG. 6.
Sxl controls the alternative splicing of its own pre-mRNA. Boxes represent exons, and lines represent introns. The male-specific exon (Sxl) is represented in red. Other exons are represented in blue (light blue for the noncoding region, and dark blue for the coding region). (A) The Sxl protein binds to poly(U) sequences in introns 2 and 3 and excludes exon L3, which contains translation stop codons, from the mature transcript. This process establishes a positive autoregulatory loop. On the other hand, Sxl binds to poly(U) sequences located at the 3′ UTR, inhibiting the translation of its own mRNA. The balance between the negative and positive posttranscriptional controls keeps the concentration of Sxl constant at levels that are nontoxic for the cell and are sufficient to regulate Sxl splicing and its target genes. (B) Model for the control of the L3 3′ splice site by SPF45 and Sxl. The splicing pattern is indicated by dashed arrows. 1, In male flies, U2AF recognizes the distal AG (AGd) and the poly(Y) tract (Py) during the first catalytic splicing step. In the second step, SPF45 recognizes the proximal AG (pAG). This AG is then, used as the site of exon ligation. 2, In female flies, Sxl binds to poly(U) sequences located at the introns adjacent to exon L3 and interacts with SPF45, blocking both proximal and distal 3′ splice site AGs from being used. Splicing then occurs between exons 2 and 4. (C) Model of regulation of exon 3 splicing based in interactions between Sxl and U2AF and U1snRNP. 1, In males, a functional spliceosome is formed and exon 3 is included in the mature transcript. 2, In females, Sxl interacts with the splicing factors U2AF and U1snRNP, bound to the 3′ and 5′ splice sites, respectively, and inhibits the formation of a functional spliceosome. As a consequence, exon 3 is not included in the mature female transcript.
FIG. 7.
FIG. 7.
The Sxl protein in different Diptera species. The comparison was done using ClustalV software and then adjusted manually. Sequences are as follows: 1, Sciara ocelaris, accession number AA019468; 2, Anopheles gambiae, accession number EAA03881; 3, Drosophila melanogaster, accession number P19339; 4, Drosophila melanogaster embryonic form (emb.), accession number A4218; 5, Drosophila subobscura, accession number Q24668; 6, Chrysomya rufifacies, accession number O97018; 7, Lucilia cuprina, accession number Q9BKK4; 8, Musca domestica, accession number O17310; 9, Ceratitis capitata, accession number O61374; and 10, Megaselia scalaris, accession number O01671. A black line is drawn above RBD1, and an orange line is drawn above RBD2. Amino acids represented in red are highly conserved (identical in six species or more). Amino acids represented in green are the ones that have equivalent biochemical properties in relation to a highly conserved amino acid. Nonconserved amino acids are represented in black. An asterisk at the bottom of the sequences indicates that in this position, the referred amino acid is conserved in all analyzed species. A colon at the bottom of the sequences indicates that in this position a highly conserved amino acid and amino acid(s) with equivalent biochemical properties are present.
FIG. 7.
FIG. 7.
The Sxl protein in different Diptera species. The comparison was done using ClustalV software and then adjusted manually. Sequences are as follows: 1, Sciara ocelaris, accession number AA019468; 2, Anopheles gambiae, accession number EAA03881; 3, Drosophila melanogaster, accession number P19339; 4, Drosophila melanogaster embryonic form (emb.), accession number A4218; 5, Drosophila subobscura, accession number Q24668; 6, Chrysomya rufifacies, accession number O97018; 7, Lucilia cuprina, accession number Q9BKK4; 8, Musca domestica, accession number O17310; 9, Ceratitis capitata, accession number O61374; and 10, Megaselia scalaris, accession number O01671. A black line is drawn above RBD1, and an orange line is drawn above RBD2. Amino acids represented in red are highly conserved (identical in six species or more). Amino acids represented in green are the ones that have equivalent biochemical properties in relation to a highly conserved amino acid. Nonconserved amino acids are represented in black. An asterisk at the bottom of the sequences indicates that in this position, the referred amino acid is conserved in all analyzed species. A colon at the bottom of the sequences indicates that in this position a highly conserved amino acid and amino acid(s) with equivalent biochemical properties are present.
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References

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