Interactive Fly, Drosophila

transformer 2


REGULATION

Targets of Activity

In addition to its role in the sex-specific control of doublesex RNA splicing insomatic tissues, the transformer-2 gene also regulates the splicing of its own transcripts in the malegerm line. The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate fourdifferent mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2products expressed in somatic tissues function to regulate the RNA splicing of the sex determinationgene doublesex, whereas products expressed in the male germ line play an unknown, but essential,role in spermatogenesis. Two alternatively spliced transformer-2 transcripts (C and E) are found only in the male germ line. These male germline-specific mRNAs differ from each other by the presence or absence of a single intron called M1.M1-containing transcripts make up a majority of transformer-2 germ-line transcriptsin wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations.Germ-line transformation experiments using a variety of reporter gene constructs demonstrate thatspecific polypeptide products of the transformer-2 gene itself normally repress M1 splicing in themale germ line. It is proposed that this autoregulatory function may serve in negative feedback control oftransformer-2 activity during spermatogenesis. The finding that transformer-2 controls multiplesplicing decisions suggests that a variety of different alternative splicing choices could be regulated bya relatively limited number of trans-acting factors (Mattox, 1991).

In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform, C, is found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving retention of intron 3. Two transcripts, A and B, code for two different isoforms that function redundantly to direct female differention and female specific doublesex pre-mRNA splicing (Mattox, 1996).

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNAprocessing. Female-specific splicing and/or polyadenylation of Doublesex pre-mRNA, the finalgene in this pathway, requires Transformer and Transformer-2 proteins. The mechanisms bywhich these proteins regulate RNA processing has not been characterized. TRA-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of DSXpre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only forfemale-specific splicing, but also for female-specific polyadenylation. These observations suggest thatTRA-2 is a positive regulator of DSX pre-mRNA processing (Hedley, 1991).

Cotransfection analyses in whichthe dsx gene and the female-specific transformer and transformer-2 complementaryDNAs were expressed in Drosophila Kc cells reveal that female-specific splicing of the DSXtranscript is positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses ofmutant constructs of DSX mRNA show that a portion of the female-specific exon sequence is required forregulation of DSX pre-messenger RNA splicing (Hoshijima, 1991).

Sex-specific alternative processing of the Doublesex pre-mRNA controls somatic sexualdifferentiation in Drosophila melanogaster. Processing in the female-specific pattern results from theutilization of an upstream terminal exon and requires the activities of both the transformer andtransformer-2 genes. Use of the more downstream male-specific terminal exons does notrequire the activities of these genes and is thus considered the default DSX-processing pattern. Transient expression of DSX pre-mRNAs in the presence or absence of tra and tra-2 geneproducts was carried out in Drosophila tissue culture cells to investigate the molecular mechanism controlling thisalternative RNA-processing decision. These studies reveal that female-specific processing of DSXpre-mRNA is controlled by TRA and TRA-2 through the positive regulation of female-specific alternative3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a540-nucleotide region from within the female exon is both necessary and sufficient for regulation. Inaddition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently offemale-specific polyadenylation. Regulated polyadenylation is obtained only in the presence ofsplicing, suggesting that activation of female-specific exon use occurs by 3'SS activation (Ryner, 1991).

Sex-specific alternative processing of doublesex precursor messenger RNA (pre-mRNA) isone of the key steps that regulates somatic sexual differentiation in Drosophila. Bytransfection analyses using dsx minigene constructs, six copies of the 13-nucleotidesequence TC(T/A)(T/A)C(A/G)ATCAACA have been identified in the female-specific fourth exon; these act as the ciselements for the female-specific splicing of DSX pre-mRNA. UV-crosslinking experiments reveal that both female-specific Transformer and Transformer-2 bind to the13-nucleotide sequences of DSX pre-mRNA. These results strongly suggest that the female-specificsplicing of DSX pre-mRNA is activated by the binding of these proteins to the 13-nucleotide sequences (Inoue, 1992).

TRA and TRA2 act byrecruiting general splicing factors to a regulatory element located downstream of a female-specific 3'splice site. Remarkably, TRA, TRA2, and members of the serine/arginine-rich (SR) family of generalsplicing factors are sufficient to commit DSX pre-mRNA to female-specific splicing, and individual SRproteins differ significantly in their ability to participate in commitment complex formation.Characterization of the proteins associated with affinity-purified complex formed on DSX pre-mRNAreveals the presence of TRA, TRA2, SR proteins, and additional unidentified components. It is concludedthat TRA, TRA2, and SR proteins are essential components of a splicing enhancer complex (Tian, 1993).

The Drosophila proteins Transformer (TRA) and Transformer2 (TRA2) regulate the sex-specificalternative splicing of Drosophila Doublesex pre-mRNA by specifically binding to a splicingenhancer (dsx repeat element, or dsxRE) located 300 nucleotides (nt) downstream from a female-specific3' splice site. The dsxRE can function as a TRA and TRA2-independentsplicing enhancer in vitro when located within 150 nucleotides of the 3' splice site. Based on therelative levels of SR proteins that bind stably to the dsxRE in the presence or absence of TRA and TRA2,it is proposed that the constitutive splicing activity of the dsxRE is mediated by its weak interactions withSR proteins and possibly other general splicing factors. In contrast, TRA and TRA2 allow the dsxRE tofunction at a distance from the intron by stabilizing the interactions between these proteins and thedsxRE (Tian, 1994).

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. 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 (SS). 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' SS 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 (Heinrichs, 1998).

To determine whether the tra/tra-2 repeat elements are essential for regulation of FRU splicing by Tra and Tra-2, a construct, fruM+FREmut, was tested in which the sequence of the conserved part of the tra/tra-2 repeat elements was changed from TCAATCAACA to GGCAGCTTAC. In construct fruM+FREmut, switching to female FRU splicing by Tra and Tra-2 is almost completely blocked, as indicated by the presence of significant amounts of male splicing product M in the presence of cotransfected tra and tra-2. In contrast, deletion of a 1-kb fragment between the repeat elements and the male-specific 5' SS, as in construct fruM+FB-M, does not affect regulation of FRU splicing by Tra and Tra-2. These findings show that the tra/tra-2 repeat elements are required for regulation of FRU splicing by Tra and Tra-2, suggesting that Tra and Tra-2 promote female-specific FRU splicing by acting through these elements (Heinrichs, 1998).

Is the tra/tra-2 repeat region in FRU sufficient to promote the activation of a 5' SS by tra/tra-2? Since the male-specific FRU 5'SS is normally unaffected by tra/tra-2, a 300-bp fragment of fru containing the tra/tra-2 repeats was inserted 4 nt upstream of the male-specific FRU 5' SS (construct fruM+REwt). Interestingly, spliced male product is detected upon cotransfection with tra/tra-2, suggesting activation of the male-specific Fru 5' SS by tra/tra-2 in this hybrid construct. Thus, the tra/tra-2 repeat elements are essential to promote the activation of a heterologous 5' SS by tra/tra-2. Lack of usage of the male-specific 5' SS in the constructs fruM+REwt and fruM+REmut in the absence of cotransfected tra/tra-2 was found to be due to the deletion of a stretch of sequence upstream of the male-specific 5' SS in these constructs. The insertion of the FRU repeat region in either orientation does not affect the usage of the male-specific FRU 5' SS in the absence of cotransfected tra/tra-2 (Heinrichs, 1998).

The Drosophila exuperantia (exu) gene encodes overlapping sex-specific, germline-dependent mRNAs. In thiswork, the structural differences between these sex-specific EXU mRNAs were determined by sequence analysis of9 ovary and 10 testis cDNAs. The transformer 2 gene functions in sex determination of female somaticcells through its role in regulating female-specific splicing of Doublesex (dsx). tra-2 isrequired in male germ cells for efficient male-specific processing of EXU RNA; in the absence of tra-2, X/Y malesproduce a new mRNA that is processed at its 3' end so that it contains sequences normally specific to thefemale 3' untranslated region. Although the processing event that requires tra-2 occurs in an untranslated regionof the EXU transcript, the isolation and characterization of a male-specific exu allele that deletes male 3'untranslated sequence indicates that this processing is biologically significant (Hazelrigg, 1994).

In male germline TRA-2 affects sex specific processing of Exuperantia and TRA2 itself, both required for spermatogenesis. Transformer 2 isoform B is necessary and sufficient for correct processing of Exuperantia pre-mRNA in the male germline. In DSX splicing, TRA and TRA2 bind directly to sequences dowstream of the female specific 3' splice site, enhancing its recognition by the general splicing machinery. In the case of TRA-2 male specific splicing, the TRA2 protein represses splicing of its own intron (Mattox, 1996).

The gene alternative testis transcripts (att) is alternatively expressed at boththe RNA and protein levels in testes and somatic tissues. The testis-specific RNA differs fromsomatic RNAs in both promoter usage and RNA processing and is dependent on the function of thetransformer 2 gene. The differences between the somatic and testis RNAs have substantialconsequences at the protein level. The somatic RNAs encode a protein with homology to themammalian Graves' disease carrier proteins. The testis RNA lacks the initiation codons used in somatictissue and encodes two different proteins. One of these begins in a testis-specific exon, uses a readingframe different from that for the somatic protein, and is completely novel. The other protein initiatestranslation in the frame of the somatic RNA at a Len CUG codon that is within the open readingframe for the somatic protein. This produces a novel truncated version of the Graves' disease carrierprotein-like protein that lacks all sequences N terminal to the first transmembrane domain (Madigan, 1996).

The Drosophila fruitless gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5' splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fruitless is regulated by Tra and Tra2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13 nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3' splice site choice in the doublesex gene. Doublesex has provided a useful model system to investigate the mechanisms of enhancer-dependent 3' splice site choice. However, little is known about enhancer dependent regulation of alternative 5 splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant Tra/Tra2 could activate the female-specific 5' splice site of fruitless. Mutational analysis has demonstrated that at least one 13 nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for doublesex, the fruRE can be replaced by a short element encompassing tandem 13 nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments show that SR proteins facilitate enhancer-dependent 5' splice site activation. It is concluded that splicing enhancers function similarly in activating regulated 5' and 3' splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5' and 3' splice sites (Lam, 2003).


transformer 2: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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