Direct repression of splicing by transformer-2

The Drosophila melanogaster sex determination factor Tra2 positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs but negatively affects the splicing of the M1 intron in tra2 pre-mRNA. Retention of the M1 intron is known to be part of a negative-feedback mechanism wherein the Tra2 protein limits its own synthesis, but the mechanism responsible for accumulation of M1-containing RNA is unknown. Here we show that the recombinant Tra2 protein specifically represses M1 splicing in Drosophila nuclear extracts. We find that the Tra2 protein binds directly to several sites in and near the M1 intron and that, when Tra2 binding is competed with other RNAs, the splicing of M1 is restored. Mapping the RNA sequences functionally required for M1 repression identified both a 34-nucleotide (nt) A/C-rich sequence immediately upstream of the M1 5' splice site and a region within the intron itself. The AC-rich sequence is largely composed of a repeated 4-nt sequence that also forms a subrepeat within the repeated 13-nt splicing enhancer elements of fru and dsx RNAs. Although required for repression, the element also enhances M1 splicing in the absence of Tra2. We propose that Tra2 represses M1 splicing by interacting with multiple sequences in the pre-mRNA and interfering with enhancer function.     

Tra2β protein is required for tissue-specific splicing of a smooth muscle myosin phosphatase targeting subunit alternative exon

Alternative splicing of the smooth muscle myosin phosphatase targeting subunit (Mypt1) exon 23 (E23) is tissue-specific and developmentally regulated and, thus, an attractive model for the study of smooth muscle phenotypic specification. We have proposed that Tra2β functions as a tissue-specific activator of Mypt1 E23 splicing on the basis of concordant expression patterns and Tra2β activation of Mypt1 E23 mini-gene splicing in vitro. In this study we examined the relationship between Tra2β and Mypt1 E23 splicing in vivo in the mouse. Tra2β was 2- to 5-fold more abundant in phasic smooth muscle tissues, such as the portal vein, small intestine, and small mesenteric artery, in which Mypt1 E23 is predominately included as compared with the tonic smooth muscle tissues, such as the aorta and inferior vena cava, in which Mypt1 E23 is predominately skipped. Tra2β was up-regulated in the small intestine postnatally, concordant with a switch to Mypt1 E23 splicing. Targeting of Tra2β in smooth muscle cells using SM22α-Cre caused a substantial reduction in Mypt1 E23 inclusion specifically in the intestinal smooth muscle of heterozygotes, indicating sensitivity to Tra2β gene dosage. The switch to the Mypt1 E23 skipped isoform coding for the C-terminal leucine zipper motif caused increased sensitivity of the muscle to the relaxant effects of 8-Br-cyclic guanosine monophosphate (cGMP). We conclude that Tra2β is necessary for the tissue-specific splicing of Mypt1 E23 in the phasic intestinal smooth muscle. Tra2β, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation.     

Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.     

Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila.

In the male germline of Drosophila the transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and att genes and autoregulates alternative splicing of its own pre-mRNA. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding TRA-2(226), which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding TRA-2(179), a protein with no known function. We have previously shown that repression of M1 splicing is dependent on TRA-2(226), suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. Here we examine this idea, by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with our hypothesis, we observe that as tra-2 gene dosage is increased, smaller proportions of TRA-2(226) mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2(226) mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of TRA-2(226). Our results suggest that autoregulation of TRA-2(226) expression in male germ cells is necessary for normal spermatogenesis.     

The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.     

前体mRNA剪接蛋白Tra2β1的抗体制备及鉴定

Tra2 β1是Tra2 β前体mRNA剪接调节蛋白家族中的一个组织分布最广、表达量最多的成员 ,它在选择性前体mRNA剪接中有调节功能 ,而在基础性剪接中不是必需的 .为便于对该蛋白功能的进一步研究 ,需要制备能特异地检测Tra2 β1蛋白的抗体 .选择包含Tra2 β1的N端 12个氨基酸的特异编码序列的Tra2 β2全长编码序列 (共 117个核苷酸 ) ,将其克隆至pGEX 3X表达载体形成GST融合基因 ,并以诱导表达和纯化的该融合蛋白为抗原免疫新西兰兔 ,获得了相应的抗体 .Western印迹和免疫细胞化学分析结果显示 ,获得的抗体能特异地检测Tra2 β1蛋白     

A Negative Feedback Mechanism Revealed by Functional Analysis of the Alternative Isoforms of the Drosophila Splicing Regulator Transformer-2

The Drosophila sex determination gene transformer-2 (tra-2) is a splicing regulator that affects the sex-specific processing of several distinct pre-mRNAs. While the tra-2 gene itself is known to produce alternative mRNAs that together encode three different TRA-2 protein isoforms, the respective roles of these isoforms in affecting individual pre-mRNA targets has remained unclear. We have generated transgenic fly strains with mutations affecting specific TRA-2 isoforms to investigate their individual roles in regulating the alternative processing of doublesex, exuperantia and tra-2 pre-mRNA. Our results indicate that in somatic tissues two different isoforms function redundantly to direct female differentiation and female-specific doublesex pre-mRNA splicing. In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform was found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving intron retention.