Sex-specific Splicing Research Articles

Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae)

doublesex ( dsx) is the bottom most gene of the sex-determination cascade of Drosophila melanogaster. The pre-mRNA of dsx splices to produce male- and female-specific transcripts which code for the male- and female-specific proteins, respectively. dsx homologues have been characterized from different (many in Diptera, two in Hypmenoptera and only one in Lepidoptera) insect species. Sex-specific splice forms of dsx pre-mRNA in all these species code for one male- and one female-specific DSX proteins, which regulate the downstream target genes responsible for sex-specific characters. In the present study we have cloned and characterized the dsx homologues from two saturniid silkmoths, Antheraea assama and Antheraea mylitta. The divergence time between Saturniidae and Bombycidae to which the domesticated silkworm, Bombyx mori belongs is estimated to be around 160.9 MY. Interestingly, the dsx pre-mRNA of these wild silkmoths sex-specifically splices to generate multiple splice variants. On the basis of their open reading frame (ORF) and conceptual translation, two female-specific (DSX F1 and DSX F2) and one male-specific (DSX M) proteins could be inferred, in both the moths. Presence or absence of a 15 bp stretch within the ORF of the two groups of female-specific transcripts resulted in the production of two distinct female-specific DSX proteins. The sex-specific DSX proteins have common amino-terminal sequence but sex-specific carboxy termini. The two female-specific DSX proteins (DSX F1 and DSX F2) share common DNA binding domain (DM domain) and oligomerization domain (OD domain) and differ only at their extreme C-termini by 21aa. Functional analysis of dsx transcripts in A. assama by dsRNA mediated knock-down resulted in complete abolition of expression of vitellogenin and hexamerin genes, the direct targets of the DSX proteins, irregular differentiation of gonads, and drastic reduction in fecundity and hatchability. Together, these results suggest the involvement of both the female-specific DSX proteins in the process of female sexual differentiation. Further, conservation of the 4th exon sequence, especially the PESS sequence responsible for the sex-specific splicing of Bmdsx in the female-specific transcripts of Aadsx and Amydsx, indicated the existence of a common mechanism of sex-specific splicing of dsx homologues in silkmoths. To our knowledge this is the first report of existence of multiple splice forms of dsx pre-mRNA encoding two female-specific DSX proteins.

Sex on the Fly

Sex on the Fly

Sex-specific and lineage-specific alternative splicing in primates

Comparative studies of gene regulation suggest an important role for natural selection in shaping gene expression patterns within and between species. Most of these studies, however, estimated gene expression levels using microarray probes designed to hybridize to only a small proportion of each gene. Here, we used recently developed RNA sequencing protocols, which sidestep this limitation, to assess intra- and interspecies variation in gene regulatory processes in considerably more detail than was previously possible. Specifically, we used RNA-seq to study transcript levels in humans, chimpanzees, and rhesus macaques, using liver RNA samples from three males and three females from each species. Our approach allowed us to identify a large number of genes whose expression levels likely evolve under natural selection in primates. These include a subset of genes with conserved sexually dimorphic expression patterns across the three species, which we found to be enriched for genes involved in lipid metabolism. Our data also suggest that while alternative splicing is tightly regulated within and between species, sex-specific and lineage-specific changes in the expression of different splice forms are also frequent. Intriguingly, among genes in which a change in exon usage occurred exclusively in the human lineage, we found an enrichment of genes involved in anatomical structure and morphogenesis, raising the possibility that differences in the regulation of alternative splicing have been an important force in human evolution.

Molecular cloning and expression analysis of Bmrbp1, the Bombyx mori homologue of the Drosophila gene rbp1

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.

The Fruitless Gene in Nasonia Displays Complex Sex-Specific Splicing and Contains New Zinc Finger Domains

The transcription factor Fruitless exerts a broad range of functions during Drosophila development, the most apparent of which is the determination of sexual behavior in males. Although fruitless sequences are found in other insect orders, little is known about fruitless structure and function outside Diptera. We have performed a thorough analysis of fruitless transcripts in the haplo-diploid wasp Nasonia vitripennis and found both sex-specific and non-sex-specific transcripts similar to those found in Drosophila. In Nasonia, however, a novel, large fruitless transcript is present in females only. Putative binding sites for sex-specific splicing factors found in Nasonia fruitless and doublesex as well as Apis mellifera doublesex transcripts were sufficient to identify a corresponding female-specific fruitless exon in A. mellifera, suggesting that similar factors in both hymenopteran species could be responsible for sex-specific splicing of both genes. Furthermore, new C(2)H(2) zinc finger domains found in Nasonia fruitless transcripts were also identified in the fruitless locus of major holometabolous insect species but not in drosophilids. Conservation of important domains and sex-specific splicing in Diptera and Hymenoptera support the hypothesis that fruitless is an ancient gene and has conserved functions in insects. Considerable divergences in other parts of the gene are expected to underlie species-specific differences and may help to explain diversity observed in insect sexual behaviors.

Sex-Specific Splicing in Drosophila: Widespread Occurrence, Tissue Specificity and Evolutionary Conservation

Many genes in eukaryotic genomes produce multiple transcripts through a variety of molecular mechanisms including alternative splicing. Alternatively spliced transcripts often encode functionally distinct proteins, indicating that gene regulation at this level makes an important contribution to organismal complexity. The multilevel splicing cascade that regulates sex determination and sex-specific development in Drosophila is a classical example of the role of alternative splicing in cell differentiation. Recent evidence suggests that a large proportion of genes in the Drosophila genome may be spliced in a sex-biased fashion, raising the possibility that alternative splicing may play a more general role in sexually dimorphic development and physiology. However, the prevalence of sex-specific splicing and the extent to which it is shared among genotypes are not fully understood. Genetic variation in the splicing of key components of the sex determination pathway is known to influence the expression of downstream target genes, suggesting that alternative splicing at other loci may also vary in functionally important ways. In this study, we used exon-specific microarrays to examine 417 multitranscript genes for evidence of sex-specific and genotype-specific splicing in 80 different genotypes of Drosophila melanogaster. Most of these loci showed sex-biased splicing, whereas genotype-specific splicing was rare. One hundred thirty-five genes showed different alternative transcript use in males vs. females. Real-time PCR analysis of 6 genes chosen to represent a broad range of biological functions showed that most sex-biased splicing occurs in the gonads. However, somatic tissues, particularly adult heads, also show evidence of sex-specific splicing. Comparison of splicing patterns at orthologous loci in seven Drosophila species shows that sexual biases in alternative exon representation are highly conserved, indicating that sex-specific splicing is an ancient feature of Drosophila biology. To investigate potential mechanisms of sex-biased splicing, we used real-time PCR to examine the expression of six known regulators of alternative splicing in males vs. females. We found that all six loci are themselves spliced sex specifically in gonads and heads, suggesting that regulatory hierarchies based on alternative splicing may be an important feature of sexual differentiation.

MTA Is an Arabidopsis Messenger RNA Adenosine Methylase and Interacts with a Homolog of a Sex-Specific Splicing Factor

N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

Doublesex and the Regulation of Sexual Dimorphism in Drosophila melanogaster: STRUCTURE, FUNCTION, AND MUTAGENESIS OF A FEMALE-SPECIFIC DOMAIN

The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSX F and DSX M) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSX F and DSX M leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSX F as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSX F interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSX F highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development.

Establishment of a Novel In Vivo Sex-Specific Splicing Assay System To Identify a trans-Acting Factor That Negatively Regulates Splicing of Bombyx mori dsx Female Exons

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.

Masculinization of XX Drosophila transgenic flies expressing the Ceratitis capitata DoublesexM isoform

The Doublesex (DSX) transcription factor regulates somatic sexual differentiation in Drosophila melanogaster. Female and male isoforms (DSXF and DSXM) are produced due to sex-specific RNA splicing. Here we show that in the distantly related dipteran Ceratitis capitata, the DSXM male-specific isoform is conserved and able to induce masculinization of both somatic and germline tissues when ectopically expressed in XX Drosophila transgenic individuals.

Specific subgroups of Fru M neurons control sexually dimorphic patterns of aggression in Drosophila melanogaster

A great challenge facing neuroscience is to understand how genes, molecules, cells, circuits, and systems interact to generate social behavior. Fruit flies (Drosophila melanogaster) offer a powerful model system to address questions of this magnitude. These animals display genetically specified, sexually dimorphic patterns of fighting behavior via sex-specific splicing of the fruitless gene. Here, we show that sexually dimorphic behavioral patterns displayed during aggression are controlled by specific subgroups of neurons expressing male forms of fruitless proteins (Fru(M)). Using the GAL4/UAS system to manipulate transformer expression, we feminized or masculinized different populations of neurons in fly nervous systems. With a panneuronal elav-GAL4 driver, male patterns of fighting behavior were transferred into females and female patterns into males. We screened 60 Gal4 lines that express the yeast transcription factor in different patterns in fly central nervous systems and found five that showed abnormal same-sex courtship behavior. The sexually dimorphic fighting patterns, however, were completely switched only in one and partially switched in a second of these lines. In the other three lines, female patterns of aggression were seen despite a switch in courtship preference. A tight correspondence was seen between Fru(M) expression and how flies fight in several subgroups of neurons usually expressing these proteins: Expression is absent when flies fight like females and present when flies fight like males, thereby beginning a separation between courtship and aggression among these neurons.

Sex-Specific Splicing of the HoneybeedoublesexGene Reveals 300 Million Years of Evolution at the Bottom of the Insect Sex-Determination Pathway

Sex-determination mechanisms vary greatly among taxa. It has been proposed that genetic sex-determination pathways evolve in reverse order from the final step in the pathway to the first step. Consistent with this hypothesis, doublesex (dsx), the most downstream gene in the Drosophila sex-determination cascade that determines most sexual phenotypes also determines sex in other dipterans and the silk moth, while the upstream genes vary among these species. However, it is unknown when dsx was recruited to the sex-determination pathway during insect evolution. Furthermore, sex-specific splicing of dsx, by which dsx determines sex, is different in pattern and mechanism between the moth and the fly, raising an interesting question of how these insects have kept the executor of sex determination while allowing flexibility in the means of execution. To address these questions, here we study the dsx gene of the honeybee Apis mellifera, a member of the most basal lineage of holometabolous insects. We report that honeybee dsx is sex-specifically spliced and that it produces both the fly-type and moth-type splicing forms, indicating that the use of different splicing forms of Dsx in controlling sexual differentiation was present in the common ancestor of holometabolous insects. Our data suggest that in ancestral holometabolous insects the female Dsx form is the default and the male form is generated by suppressing the splicing of the female form. Thus, it is likely that the dsx splicing activator system in flies, where the male form is the default, arose during early dipteran evolution.

The gene doublesex of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

The doublesex (dsx) gene of several Anastrepha species was isolated and characterised. Its molecular organisation was found to be the same in all the species examined. This gene is composed of four exons: Exons 1 and 2 are common to both sexes, exon 3 is female specific, and exon 4 is male specific. It codes for both the female DsxF and male DsxM proteins, corresponding to the sex-specific splicing product of its primary transcript; male-specific splicing is the default mode. A comparison of the Dsx proteins of different Anastrepha species with those of other insects showed them to be very similar. Molecular evolutionary analysis (both at the nucleotide and amino acid levels) of dsx in different insects revealed a topology in good agreement with their owners' taxonomic relationships. The great majority of the nucleotide changes detected in the dsx gene of the analysed species were significantly synonymous, evidence that strong purifying selection has acted on dsx so that the functional structure of the Dsx proteins is preserved. However, the common region of DsxF and DsxM proteins appeared to be the main target for selection acting upon the long-term evolution of gene dsx.

The Sex-Determination Genes fruitless and doublesex Specify a Neural Substrate Required for Courtship Song

SummaryCourtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1–6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10–13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (FruM), we show a critical requirement for the male isoform of dsx (DsxM), alongside FruM, in the specification of courtship song. Therefore, although FruM expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without DsxM, the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.

Female-specific insect lethality engineered using alternative splicing

The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters.

The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

Fruitless regulates aggression and dominance in Drosophila

When competing for resources, two Drosophila melanogaster flies of the same sex fight each other. Males and females fight with distinctly different styles, and males but not females establish dominance relationships. Here we show that sex-specific splicing of the fruitless gene plays a critical role in determining who and how a fly fights, and whether a dominance relationship forms.

Concentration dependent selection of targets by an SR splicing regulator results in tissue-specific RNA processing

The splicing factor Transformer-2 (Tra2) is expressed almost ubiquitously in Drosophila adults, but participates in the tissue-specific regulation of splicing in several RNAs. In somatic tissues Tra2 participates in the activation of sex-specific splice sites in doublesex and fruitless pre-mRNAs. In the male germline it affects splicing of other transcripts and represses removal of the M1 intron from its own pre-mRNA. Here we test the hypothesis that the germline specificity of M1 repression is determined by tissue-specific differences in Tra2 concentration. We find that Tra2 is expressed at higher levels in primary spermatocytes of males than in other cell types. Increased Tra2 expression in other tissues reduces viability in a manner consistent with known dose-dependent effects of excessive Tra2 expression in the male germline. Somatic cells were found to be competent to repress M1 splicing if the level of Tra2 transcription was raised above endogenous concentrations. This suggests not only that M1 repression is restricted to the germline by a difference in Tra2 transcription levels but also that the protein's threshold concentration for M1 regulation differs from that of doublesex and fruitless RNAs. We propose that quantitative differences in regulator expression can give rise to cell-type-specific restrictions in splicing.

The Gene doublesex of the Fruit Fly Anastrepha obliqua (Diptera, Tephritidae)

The gene doublesex of Anastrepha obliqua is composed of four instead of the usual six exons. It is transcribed in both sexes and its primary transcript undergoes sex-specific splicing, producing female Dsx(F) and male Dsx(M) proteins, which have in common the amino-terminal region but which differ at the carboxyl-terminal region.

Inventing a sex-specific gene: a conserved role of DMRT1 in teleost fishes plus a recent duplication in the medaka Oryzias latipes resulted in DMY.

DMY, the first sex-determining gene to be described in a nonmammal vertebrate was recently characterized in the medaka fish (Oryzias latipes). It is homologous to DMRT1, a conserved gene of the sex determination cascade in vertebrates. We have checked the near complete genomes of two other percomorph fishes, Tetraodon nigroviridis and Takifugu rubripes, for supplementary homologs of DMRT1 and DMY. We also compared the new gene, DMY, to its homolog DMRT1 from all available vertebrates. Finally, we found evidence for sex-specific expression and alternative splicing of the homolog from T. nigroviridis. Our results show that DMY is a recent duplicate of DMRT1 in the medaka. Its role in sex determination was not acquired through an acceleration of evolutionary rates, but by translocation to the Y chromosome and possibly changes at key positions.