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

Gene name - transformer 2

Synonyms -

Cytological map position - 51B4--51B6

Function - mRNA splicing factor

Keyword(s) - Sex determination, Splicing factor

Symbol - tra2

FlyBase ID: FBgn0003742

Genetic map position - 2-70

Classification - Serine/Arginine motif, RNA recognition motif

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Li, J. and Handler, A. M. (2017). Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing Drosophila, Drosophila suzukii. Sci Rep 7(1): 12363. PubMed ID: 28959033
Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9-directed repair gene-editing. Ds-tra-2ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 ° C, but at higher restrictive temperatures (26 to 29 ° C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-Ds-tra-2ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

The splicing of Doublesex pre-messenger RNA has become the primary model for the process of alternative splicing of RNAs in all higher organisms. Essential to this splicing is Transformer 2, a protein containing arginine/serine rich domains involved in protein-protein interaction. This overview will outline the complex process of splicing, highlighting the specific importance of alternative splicing, with special attention paid to the role of Transformer 2 protein.

In the most general terms, splicing is the removal of an intron, a specified portion of the nucleotide chain, from between two flanking exons (portions of the pre-messenger RNA that code for proteins). The process is regulated by certain proteins called splice factors, and depends upon the successful construction of splice machinery, in particular a structure known as a spliceosome, a multiprotein complex that is assembled step by step during the splicing process. Alternative splicing generates alternative messenger RNA species, depending on the pattern of intron removal. For example, by alternative splicing in which exons are skipped, unwanted exons can be excluded altogether from mRNA species coding for a particular polypeptide chain, thus generating a functionally alternative protein.

Imagine the intron, a stretch of nucleotides to be removed, bracketed between two exons, one upstream and one downstream. In the case of Doublesex pre-mRNA, the intron to be removed lies between exons 3 and 4. When this particular intron is productively removed, a female specific Doublesex mRNA is produced. The female specific Doublesex mRNA, as its name implies, codes for a female specific Doublesex protein that will result in the development of female flies. When the intron is not productively removed, the exon 3 becomes spliced instead to a downstream exon 5, resulting in the production of a male specific Doublesex mRNA, and the consequent development of male flies.

This all important splice event is regulated by two proteins: Transformer and Transformer 2. Splicing of TRA and TRA2 pre-mRNA is regulated by the splice factor Sex lethal, the upstream master regulator of sex determination in Drosophila. Sex lethal assures that TRA and TRA2 proteins are present only in females. TRA and TRA2 proteins act subsequently on Doublesex pre-mRNA to regulate splicing that will result in a female specific Doublesex mRNA. In the absence of TRA and TRA2, a male specific Doublesex mRNA is attained, guaranteeing a male developmental fate.

How exactly do TRA and TRA2 accomplish the splicing of DSX pre-mRNA? To answer this question, it is necessary to look more closely at what happens biochemically during splicing. Critical to this process is the initial recognition of the two ends of the intron. The left hand, upstream end, known as the 5' splice site, is first recognized by U1 small nuclear ribonucleoprotein (snRNP), a multiprotein complex consisting of among others, an A subunit, a C subunit and a 70 kD ribonucleoprotein with an RNA recognition motif and two arginine-serine rich RS motifs (for more information on Drosophila U1 snRNP, see sans fille). The right hand downstream end, known as the 3' splice site is recognized by U2AF65, a protein with three RNA recognition motifs and an arginine-serine rich RS motif. Another protein (U2AF35) that interacts with U2AF65 must also assembling at the 3 ' splice site (Chabot, 1996).

At this point, after the proteins listed above are poised for action, another group of proteins (called SR proteins) become involved. The human SR protein family comprises at least six members, designated SRp75, Srp55, SRp40, SRp30a, SRP30b and SRp20; these are conserved from Drosophila to humans. In Drosophila, only the SR proteins RBP1 (homologous to SRp20) and B52 (homologous to SRp55) have been cloned (Heinrichs, 1995 and references). The SR proteins share a characteristic domain structure consisting of an N-terminal RRM type RNA binding domain known to be involved in RNA binding and a C-terminal serine-arginine SR domain. What happens is that the SR proteins act as a bridge between U1, associated with the 5' splice site and U2AF65/U2AF35, assembled at the 3' splice site. Essentially the SR proteins are involved in the assembly of the spliceosome. It is thought that SR proteins can regulate alternative splicing in many pre-mRNAs, but in DSX mRNA splicing the principle regulators are TRA and TRA2 (Chabot, 1996). Once the spliceosome is assembled, removal of the intron is achieved by a complex program involving additional steps (Lamond, 1994) that will not be described here.

So far this overview has focused on the intron stretched between upstream and downstream exons of DSX pre-mRNA. A closer look at the middle of exon 4 is now in order, the site from which TRA and TRA2 act. Here is found one of the wonders of nucleic acid: a sequence coding for a protein can have hidden in its triplet codes, a region with an altogether different function, in this case a sequence that binds splicing regulators and influences the intron splicing event taking place upstream from its hidden message. The message inside exon 4 is a highly structured region called the dsx repeat element (dsxRE); it contains six copies of a 13-nucleotide repeat sequence. Between repeat elements 5 and 6 of the dsxRE is another element, the purine-rich enhancer (PRE). Thus the dsxRE consists of two types of regulatory sequences, the repeat elements R1-6, and the PRE (Lynch, 1995 and references).

TRA2 binds to repeat elements R2-5 with significant specificity. In contrast, the SR proteins bind with very little, although measurable, specificity. TRA does not bind specifically to the dsxRE. The specificity of binding for the combination of TRA and an SR protein is 10-15 times greater than that observed with either protein alone. Moreover, the addition of TRA2 to TRA plus an SR protein further increases the specificity of the TRA + SR complex by two to threefold (Lynch, 1996). It is thought that the SR protein RBP1 binds the purine-rich enhancer (PRE) in carrying out its role as a catalyst for assembly of TRA and TRA2, which interact with the repeat elements in the dsxRE (Heinrichs, 1995). TRA, TRA2 and RBP1 serve to facilitate the assembly of a functional spliceosome associated with the intron between exons 3 and 4.

The 3' splice site of the DSX intron is actually thought to be suboptimal, that is, without help from the dsxRE, unaided splicing would favor the male outcome: exon 4 of the DSX gene would be passed over and splicing would occur between exons 3 and 5 of the DSX pre-mRNA. The splice enhancer is utilized in the females, and is functional because of the presence of TRA and TRA2 (Tian, 1994). TRA's role is special in that splicing of the TRA pre-mRNA of Drosophila is regulated by Sex-lethal-dependent 3' splice site blockage, generating a female specific TRA protein (Sosnowski, 1994). Thus presence of female specific TRA, but not TRA2 which does not differ between males and females, phenotypically distinguishes males and females, and the role of TRA2 is to lend specificity to joining of 3' and 5' splice junctions.

For information on the role of Sex Lethal in TRA pre-mRNA, see Sex lethal. For information on the biological role of Doublesex, see Doublesex.


cDNA clone length - 1173

Bases in 5' UTR - 223

Exons - 7

Bases in 3' UTR - 402


The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2 products expressed in somatic tissues (RNA Types A and B) function to regulate the RNA splicing of the sex determination gene doublesex. The two somatic line variants are initiated from the same start site, but differ in the presence or absence of a third exon. Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line (RNA Types C and E). These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1, found between exons 3 and 4. M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations. An additional transcript (Type D) codes for a C-terminally truncated protein (Mattox, 1990 and 1991).

In the male germline, where tra-2 is essential for spermatogenesis, a single isoform (isoform B, coded for by RNA type E) is found that uniquely performs all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving retention of intron 3 (Mattox, 1996).

As previously stated, the Drosophila transformer-2 gene is required for female sex determination in somatic cells and for spermatogenesis in male germ cells. Two transcripts are detected in males and females; they differ in their abundance and in the presence (minor transcript Tmin) or absence (major transcript Tmaj) of one exon (Exon 3). Transcript Tmaj codes for isoform A (264 amino acids) protein and transcript Tmin codes for isoform B protein (226 amino acids). Two other transcripts are present only in male germ cells. One of these is rare (msTmin) and represents a spliced form (coding for isoform B) of the other and more abundant transcript (msTmaj - a non-productively spliced mRNA species), differing from msTmin in the presence or absence of intron 3. The transcript Tmaj encodes a protein of 264 amino acids, whereas transcripts Tmin encodes isoform B that is truncated at the N-terminus. All proteins contain a stretch of approximately 90 amino acids, the ribonucleoprotein motif (RNP motif), which shows similarity to a variety of different ribonucleoproteins. Transformation studies reveal that a cDNA corresponding to the transcript Tmaj can provide all the functions for female sex determination and male fertility. Surprisingly, a cDNA corresponding to the transcript msTmaj could only supply some female sex-determining function, but was unable to restore fertility in mutant males. Sequence analysis of two temperature-sensitive mutations provides evidence that the RNP motif represents an important functional domain of the TRA-2 protein (Amrein, 1990).

Expression of either the A or B isoform alone is sufficient for correct sexual differention in the soma. Both isoform A and B are normally synthesized in the soma, while isoform B is normally synthesized in the male germline. Isoform B autoregulates its synthesis in the male germline by blocking a splicing pathway leading to the mRNA that encodes it. Isoform B is necessary and sufficient for correct processing of Exuperantia pre-mRNA in the male germline (Mattox, 1996).

Amino Acids - 264 (type A), 226 (type B), 179 (Mattox, 1990).

Structural Domains

The TRA2 protein central domain spans approximately 80 amino acids and shows similarities to a family of proteins that bind single-stranded nucleic acids. On either side of the RNA recognition motif are arginine/serine motifs similar to those found in SR proteins (Amrein, 1988 and Goralski, 1989).

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

date revised: 10 Mar 97 

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