Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.     

Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation

Sex-specific alternative processing of the doublesex (dsx) pre-mRNA controls somatic sexual differentiation in Drosophila melanogaster. Processing in the female-specific pattern results from the utilization of an upstream 3'-terminal exon and requires the activities of both the transformer (tra) and transformer-2 (tra-2) genes. Use of the more downstream male-specific terminal exons does not require the activities of these genes and is thus considered the default dsx-processing pattern. Here, we used transient expression of dsx pre-mRNAs in the presence or absence of tra and tra-2 gene products in Drosophila tissue culture cells to investigate the molecular mechanism controlling this alternative RNA-processing decision. These studies reveal that female-specific processing of dsx pre-mRNA is controlled by tra and tra-2 through the positive regulation of female-specific alternative 3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a 540-nucleotide region from within the female exon is both necessary and sufficient for regulation. In addition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently of female-specific polyadenylation. Regulated polyadenylation was obtained only in the presence of splicing, suggesting that activation of female-specific exon use occurs by 3'SS activation.     

A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans

The normal sexes of Caenorhabditis elegans are the self-fertilizing hermaphrodite (XX) and the male (XO). The autosomal gene tra-1 is a major switch gene controlling sexual phenotype. Mutant phenotypes of 43 loss-of-function (lf) tra-1 alleles and 22 gain-of-function (gf) tra-1 alleles are described and discussed. The tra-1(lf) alleles are recessive and, in general, masculinizing. The most severe mutations (such as seven out of eight identified amber alleles) can transform XX animals into fertile males. These mutations have little effect on XO animals (which are male already) but lead to some abnormalities in XO gonadal development, indicating that tra-1 has functions in normal development of both sexes, although its major function is confined to the XX hermaphrodite. Weaker tra-1(lf) alleles lead to incomplete masculinization of XX animals, resulting in a variety of intersexual phenotypes. the tra-1(gf) alleles are dominant and have an opposite, feminizing effect. Six out of 22 can transform XO animals into fertile females or hermaphrodites, whereas the remainder cause partial feminization. All 22 transform XX animals into fertile females. Limited intragenic mapping indicates that the gene is large and that gf alleles map to a location different from lf alleles. The results suggest that the tra-1 gene has several roles in wild-type sexual development. First, tra-1 activity dictates female, as opposed to male, development in all nongonadal tissues of XX animals. Second, tra-1 activity dictates female development in the somatic gonad of XX animals. Third, a high level of tra-1 activity may act to inhibit spermatogenesis in the XX germ line, thereby assisting the switch from spermatogenesis to oogenesis in the hermaphrodite. These three functions are all feminizing and specific to the XX animal; the gene also has minor functions in the XO animal, which are to assist normal male somatic gonad development and to promote abundant spermatogenesis in males. A low level of both spermatogenesis and oogenesis can occur in the absence of tra-1 activity. Both the function and the regulation of this gene must be complex.     

Sexually dimorphic neurons of the terminalia of Drosophila melanogaster: II. Sex-specific axonal arborizations in the central nervous system

In order to understand how the peripheral nervous system in male and female flies differ, I have determined the projection pattern of sensory neurons that innervate the sex-specific adult terminalia, the genitalia and analia, of Drosophila melanogaster. In the adult male and female fly, mechanosensory bristles arranged on the external terminalia are innervated by sensory neurons that arborize in the abdominal ganglion. The distribution of axonal arbors differs between males and females. In males, sensory neurons terminate in a greater variety of patterns over a larger area of neuropil than those in females. Mutations in a sex-determining gene (transformer-2, tra-2,) which regulates the development of secondary sexual characteristics in somatic tissues, cause female flies to be transformed into phenotypic males. The sensory neurons of the terminalia are also transformed leading them to arborize appropriately for male neurons.     

Negative regulation of male development in Caenorhabditis elegans by a protein–protein interaction between TRA-2A and FEM-3

The tra-2 gene of the nematode Caenorhabditis elegans encodes a predicted membrane protein, TRA-2A, that promotes XX hermaphrodite development. Genetic analysis suggests that tra-2 is a negative regulator of three genes that are required for male development: fem-1, fem-2, and fem-3. We report that the carboxy-terminal region of TRA-2A interacts specifically with FEM-3 in the yeast two-hybrid system and in vitro. Consistent with the idea that FEM-3 is a target of negative regulation, we find that excess FEM-3 can overcome the feminizing effect of tra-2 and cause widespread masculinization of XX somatic tissues. In turn, we show that the masculinizing effects of excess FEM-3 can be suppressed by overproduction of the carboxy-terminal domain of TRA-2A. A FEM-3 fragment that retains TRA-2A-binding activity can masculinize fem-3(+) animals, but not fem-3 mutants, suggesting that it is possible to release and to activate endogenous FEM-3 by titrating TRA-2A. We propose that TRA-2A prevents male development by interacting directly with FEM-3 and that a balance between the opposing activities of TRA-2A and FEM-3 determines sex-specific cell fates in somatic tissues. When the balance favors FEM-3, it acts through or with the other FEM proteins to promote male cell fates.     

Preface to the Special Issue “Genes,Circuits, and Behaviors: Coming of Age”

In order to understand how the peripheral nervous system in male and female flies differ, I have determined the projection pattern of sensory neurons that innervate the sex-specific adult terminalia, the genitalia and analia, of Drosophila melanogaster. In the adult male and female fly, mechanosensory bristles arranged on the external terminalia are innervated by sensory neurons that arborize in the abdominal ganglion. The distribution of axonal arbors differs between males and females. In males, sensory neurons terminate in a greater variety of patterns over a larger area of neuropil than those in females. Mutations in a sex-determining gene (transformer-2, tra-2) which regulates the development of secondary sexual characteristics in somatic tissues, cause female flies to be transformed into phenotypic males. The sensory neurons of the terminalia are also transformed leading them to arborize appropriately for male neurons.     

The ovo locus is required for sex-specific germ line maintenance in Drosophila

Mutations at the ovo locus result in a defective female germ line. The male germ line is not affected. Adult females homozygous for loss-of-function alleles have no germ line stem cells. The sex-specific phenotype is evident at late blastoderm and early gastrula stages when the pole cells of embryos homozygous for a loss-of-function allele begin to die. This is the only zygotically acting gene known that is required specifically for embryonic germ line survival. Females heterozygous for dominant alleles or homozygous for alleles reducing gene activity exhibit a range of defects in oogenesis. We have mapped the ovo locus to position 4E1-2 of the salivary gland X chromosome by using a set of cytologically visible deletions.     

Mammalian sex chromosomes III. Activity of pseudoautosomal steroid sulfatase enzyme during spermatogenesis inMus musculus

Parallel to the inactivation of the X chromosome in somatic cells of female, the male X in mammals is rendered inactive during spermatogenesis. Pseudoautosomal genes, those present on the X-Y meiotically pairable region of male, escape inactivation in female soma. It is suggested, but not demonstrated, that they may also be refractory to the inactivation signal in male germ cells. We have assayed activity of the enzyme steroid sulfatase, product of a pseudoautosomal gene, in testicular cells of the mouse and shown its presence in premeiotic, meiotic (pachytene), and postmeiotic (spermatid) cell types. It appears that, as in females, pseudoautosomal genes may escape inactivation in male germ cells also.     

Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish.

Although mechanisms of sex differentiation have been studied intensely in mammals, insects, and worms, little is known about this process in lower vertebrates. To establish a marker for female gonad differentiation in zebrafish, we generated a transgenic line in which 412 bp from the promoter and 5' mRNA leader of the female-specific zebrafish zona pellucida gene zpc are fused to the coding region of green fluorescent protein (GFP). The zpc0.5:GFP transgene is expressed exclusively in oocytes, starting from the onset of female-specific differentiation, and closely resembles the expression pattern of the wild-type zpc. Strong GFP expression persists throughout oogenesis and is visible through the body wall of females. We have also characterized a putative upstream factor of zpc, FIGalpha, and show that distribution of FIGalpha RNA is compatible with its postulated role in the regulation of zpc. The zpc0.5:GFP transgenic line described here will be useful for studying oocyte development and the mechanisms that determine sex-specific gene expression in the zebrafish. It is also the first promoter characterized to date to drive stable and efficient expression specifically in the zebrafish female germline.     

Identification of in vivo mRNA targets of GLD-1, a maxi-KH motif containing protein required for C. elegans germ cell development

Caenorhabditis elegans GLD-1, a KH motif containing RNA-binding protein of the GSG/STAR subfamily, controls diverse aspects of germ line development, suggesting that it may have multiple mRNA targets. We used an immunoprecipitation/subtractive hybridization/cloning strategy to identify 15 mRNAs that are putative targets of GLD-1 binding and regulation. For one target, the rme-2 yolk receptor mRNA, GLD-1 acts as a translational repressor to spatially restrict RME-2 accumulation, and thus yolk uptake, to late-stage oocytes. We found that GLD-1 binds sequences in both 5' coding and the 3' untranslated region of rme-2 mRNA. Initial characterization of the other 14 targets shows that (1) they are coexpressed with GLD-1; (2) they can have mutant/RNA-mediated interference depletion phenotypes indicating functions in germ line development or as maternal products necessary for early embryogenesis; and (3) GLD-1 may coregulate mRNAs corresponding to functionally redundant subsets of genes within two gene families. Thus, a diverse set of genes have come under GLD-1-mediated regulation to achieve normal germ line development. Previous work identified tra-2 as a GLD-1 target for germ line sex determination. Comparisons of GLD-1-mediated translational control of rme-2 and tra-2 suggests that the mechanisms may differ for distinct target mRNA species.