Tra2 Proteins Research Articles

Regulatory Interplay Between SR Proteins Governs CLK1 Kinase Splice Variants Production.

The CLK1 kinase phosphorylates SR proteins to modulate their splicing regulatory activity. Skipping of alternative exon 4 on the CLK1 pre-mRNA produces a CLK1 variant lacking the catalytic site. Here, we aimed to understand how various SR proteins integrate into the regulatory program that controls CLK1 exon 4 splicing. Previously, we observed that the depletion of SRSF10 promoted the inclusion of CLK1 exon 4. Using expression of tagged proteins and CRISPR/Cas9-mediated knockouts in HCT116 cells, we now identify TRA2b, TRA2a, SRSF4, SRSF5, SRSF7, SRSF8 and SRSF9 as activators of exon 4 inclusion. In contrast, SRSF3, SRSF10 and SRSF12 elicit exon 4 skipping. Using CRISPR/dCas13Rx and RNA immunoprecipitation assays, we map an enhancer in exon 4 interacting with TRA2b. Notably, CLK1 kinase inhibitors antagonized the repressor activity of HA-SRSF10, HA-SRSF12 and HA-SRSF3. Our results suggest that CLK1 exon 4 inclusion is determined primarily by a balance between the activities of TRA2 proteins and CLK-phosphorylated SRSF3. CLK-phosphorylated SRSF10 and SRSF12 would interact with TRA2 proteins to prevent their enhancer activity, allowing SRSF3 to enforce exon 4 skipping more efficiently. Our study provides insight into the complex regulatory network controlling the alternative splicing of CLK1, which uses CLK1-mediated phosphorylation of SR proteins to regulate the inclusion of catalytic exon 4 in CLK1 transcripts.

Screening and Verification of Photosynthesis and Chloroplast-Related Genes in Mulberry by Comparative RNA-Seq and Virus-Induced Gene Silencing.

Photosynthesis is one of the most important factors in mulberry growth and production. To study the photosynthetic regulatory network of mulberry we sequenced the transcriptomes of two high-yielding (E1 and E2) and one low-yielding (H32) mulberry genotypes at two-time points (10:00 and 12:00). Re-annotation of the mulberry genome based on the transcriptome sequencing data identified 22,664 high-quality protein-coding genes with a BUSCO-assessed completeness of 93.4%. A total of 6587 differentially expressed genes (DEGs) were obtained in the transcriptome analysis. Functional annotation and enrichment revealed 142 out of 6587 genes involved in the photosynthetic pathway and chloroplast development. Moreover, 3 out of 142 genes were further examined using the VIGS technique; the leaves of MaCLA1- and MaTHIC-silenced plants were markedly yellowed or even white, and the leaves of MaPKP2-silenced plants showed a wrinkled appearance. The expression levels of the ensiled plants were reduced, and the levels of chlorophyll b and total chlorophyll were lower than those of the control plants. Co-expression analysis showed that MaCLA1 was co-expressed with CHUP1 and YSL3; MaTHIC was co-expressed with MaHSP70, MaFLN1, and MaEMB2794; MaPKP2 was mainly co-expressed with GH9B7, GH3.1, and EDA9. Protein interaction network prediction revealed that MaCLA1 was associated with RPE, TRA2, GPS1, and DXR proteins; MaTHIC was associated with TH1, PUR5, BIO2, and THI1; MaPKP2 was associated with ENOC, LOS2, and PGI1. This study offers a useful resource for further investigation of the molecular mechanisms involved in mulberry photosynthesis and preliminary insight into the regulatory network of photosynthesis.

Molecular characterization of transformer‐2 gene in the mud crab Scylla paramamosain and its putative role in sexual development

Transformer-2 (Tra-2) is an mRNA splicing factor that acts in concert with Tra to regulate the female sexual development in Drosophila melanogaster. In decapods, as no Tra homologues have been identified, the role of Tra-2 in sexual development is more often discussed accordingly. In the present study, the full-length cDNA of Tra-2 of the mud crab Scylla paramamosain (SpTra-2) was cloned and characterized. The deduced amino acid sequence of SpTra-2 contained an RNA-recognition motif (RRM) flanking by two RS-rich regions, which are the hallmarks of the Tra-2 proteins. The SpTra-2 mRNA was highly abundant in the hepatopancreas, muscles and androgenic glands of the male crabs, implying a potential role in male sexual development. The hypothesis was supported by the RNAi experiments, finding that the injection of SpTra-2 dsRNA in vivo led to a decreased expression of SpIAG, a key gene in crustacean male sexual differentiation. The relationship between SpTra-2 and SpIAG was further affirmed by eyestalk ablation (ESA) experiments, which caused SpTra-2 expression earlier than SpIAG expression in both androgenic glands and hepatopancreas. In addition, treatment with SpTra-2 dsRNA also reduced the mRNA level of SpSxl, but had no effects on SpDmrt expression, suggesting the sex determination system in S. paramamosain is different from that in D. melanogaster.

Two of the three Transformer-2 genes are required for ovarian development in Aedes albopictus

In Drosophila melanogaster, transformer-2 (tra2) plays an essential role in the sex-specific splicing of doublesex (dsx) and fruitless (fru), two key transcription factor genes that program sexual differentiation and regulate sexual behavior. In the present study, the sequences and expression profiles of three tra2 (Aalbtra2) genes in the Asian tiger mosquito, Aedes albopictus (Ae. albopictus) were characterized. Phylogenetic analysis revealed that these paralogs resulted from two duplication events. The first occurred in the common ancestor of Culicidae, giving rise to the tra2-α and tra2-β clades that are found across divergent mosquito genera, including Aedes, Culex, and Anopheles. The second occurred within the tra2-α clade, giving rise to tra2-γ in Ae. albopictus. In addition to the conserved RNA recognition motif (RRM), arginine-rich/serine-rich regions (RS domains) and a linker region, a glycine-rich region located between the RRM and RS2 was observed in Tra2-α and Tra2-γ of Ae. albopictus that has not yet been described in the Tra2 proteins of dipteran insects. Quantitative real-time PCR detected relatively high levels of transcripts from all three tra2 paralogs in 0–2 h embryos, suggesting maternal deposition of these transcripts. All three Aalbtra2 genes were highly expressed in the ovary, while Aalbtra2-β was also highly expressed in the testis. RNAi-mediated knockdown of any or all Aalbtra2 genes did not result in an obvious switch of the sex-specificity in dsx and fru splicing in the whole-body samples. However, knockdown of transcripts from all three tra2 genes significantly reduced the female isoform of dsx mRNA and increased the male isoform of the dsx mRNA in both the ovary and the fat body in adult females. Furthermore, knockdown of either Aalbtra2-α or Aalbtra2-γ or all three Aalbtra2 led to a decrease in ovariole number and ovary size after a blood meal. Taken together, these results indicate that two of the three tra2 genes affect female ovarian development.

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2β, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain–RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain–RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the ‘compensatory’ mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

Increased expression levels of the RNA splicing regulator Transformer2β (abbreviated Tra2β) have been reported in several types of cancer. Recent work has revealed an intimate cross-regulation between Tra2β and the highly similar Tra2α protein in human breast cancer cells, though these two proteins are encoded by separate genes created by a gene duplication that occurred over 500 million years ago. This cross-regulation involves splicing control of a special class of exons, called poison exons. Down-regulation of Tra2β reduces splicing inclusion of a poison exon in the mRNA encoding Tra2α, thereby up-regulating Tra2α protein expression. This buffers any splicing changes that might be caused by individual depletion of Tra2β alone. Discovery of this cross-regulation pathway, and its by-pass by joint depletion of both human Tra2 proteins, revealed Tra2 proteins are essential for breast cancer cell viability, and led to the identification of important targets for splicing control. These exons include a critical exon within the checkpoint kinase 1 (CHK1) gene that plays a crucial function in the protection of cancer cells from replication stress. Breast cancer cells depleted for Tra2 proteins have reduced CHK1 protein levels and accumulate DNA damage. These data suggest Tra2 proteins and/or their splicing targets as possible cancer drug targets.

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons.

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.

Tra2 protein biology and mechanisms of splicing control.

Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.

Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans.

The ubiquitin-binding RPN-10 protein serves as a ubiquitin receptor that delivers client proteins to the 26S proteasome. Although ubiquitin recognition is an essential step for proteasomal destruction, deletion of the rpn-10 gene in yeast does not influence viability, indicating redundancy of the substrate delivery pathway. However, their specificity and biological relevance in higher eukaryotes is still enigmatic. We report herein that knockdown of the rpn-10 gene, but not any other proteasome subunit genes, sexually transforms hermaphrodites to females by eliminating hermaphrodite spermatogenesis in Caenorhabditis elegans. The feminization phenotype induced by deletion of the rpn-10 gene was rescued by knockdown of tra-2, one of sexual fate decision genes promoting female development, and its downstream target tra-1, indicating that the TRA-2-mediated sex determination pathway is crucial for the Delta rpn-10-induced sterile phenotype. Intriguingly, we found that co-knockdown of rpn-10 and functionally related ubiquitin ligase ufd-2 overcomes the germline-musculinizing effect of fem-3(gf). Furthermore, TRA-2 proteins accumulated in rpn-10-defective worms. Our results show that the RPN-10-mediated ubiquitin pathway is indispensable for control of the TRA-2-mediated sex-determining pathway.

Cloning and Alternative Splicing Analysis of <italic>Bombyx mori</italic> Transformer-2 Gene using Silkworm EST Database

We have identified Bombyx mori transformer-2 gene (Bmtra-2) cDNA by blasting the EST database of B. mori. It was expressed in the whole life of the male and female silkworm and was observed as a band of 1.3 kb by Northern blot analysis. By comparing corresponding ESTs to the Bmtra-2 DNA sequence, it was revealed that there were eight exons and seven introns, and all splice sites of exons/introns conformed to the GT/AG rule. Bmtra-2 pre-mRNA can produce multiple mRNAs encoding six distinct isoforms of BmTRA-2 protein using an alternative splicing pathway during processing. Six types of Bmtra-2 cDNA clones were identified by reverse transcription-polymerase chain reaction. All isoforms of BmTRA-2 protein contain two arginine/serine-rich domains and one RNA recognition motif, showing striking organizational similarity to Drosophila TRA-2 proteins.

Up-regulation of the ubiquitous alternative splicing factor Tra2β causes inclusion of a germ cell-specific exon

We have discovered a new exon of the homeodomain-interacting kinase HipK3 that incorporates a premature stop codon and is included only in the human testis. To investigate this, we tested the effects of transfecting cells with green fluorescent protein fusions of RNA-binding proteins implicated in spermatogenesis using a novel assay based on multi-fraction fluorescence-activated cell sorting (MF-FACS). This allows the effect of a controlled titration of any splicing factor on the splicing of endogenous genes to be studied in vivo. We found that Tra2beta recapitulates testis-specific splicing of endogenous HipK3 in a concentration-dependent manner and binds specifically to a long purine-rich sequence in the novel exon. This sequence was also specifically bound by hnRNP A1, hnRNP H, ASF/SF2 and SRp40, but not by 9G8. Consistent with these observations, in vitro studies showed that this sequence shifts splicing to a downstream 5' splice site within a heterologous pre-mRNA substrate in the presence of Tra2beta, ASF/SF2 and SRp40, whereas hnRNP A1 specifically inhibits this choice. By mutating the purine-rich sequence in the context of the HipK3 gene, we also show that it is the major determinant of Tra2beta- and hnRNP A1-mediated regulation. Tra2 is essential for sex determination and spermatogenesis in flies, and Tra2beta protein was most highly expressed in testis out of six mouse tissues, whereas hnRNP A1 is down-regulated during germ cell development. Therefore, our data imply an evolutionarily conserved role for Tra2 proteins in spermatogenesis and suggest that an elevated concentration of Tra2beta may convert it into a tissue-specific splicing factor.

Functions of SR and Tra2 Proteins in Pre-mRNA Splicing Regulation

Functions of SR and Tra2 Proteins in Pre-mRNA Splicing Regulation

SR proteins are ‘locators’ of the RNA splicing machinery

B.R.G. and K.J.H. were supported by the Jane Coffin Childs Memorial Fund for Medical Research and T.M. by the NIH.

Human Tra2 Proteins Are Sequence-Specific Activators of Pre-mRNA Splicing

The RNA-binding protein Tra2 is an important regulator of sex determination in Drosophila. Recently, two mammalian Tra2 homologs of unknown function have been described. Here, we show that human Tra2 proteins are present in HeLa cell nuclear extracts and that they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. Indeed, both purified proteins bound preferentially to RNA sequences containing GAA repeats, characteristic of many enhancer elements. Neither Tra2 protein functioned in constitutive splicing in vitro, but both activated enhancer-dependent splicing in a sequence-specific manner and restored it after inhibition with competitor RNA. Our findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns.

Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2.

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. Here we report that 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. 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' splice site 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.

A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.

Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila. Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2 alpha, a human homologue of tra-2. Two alternative types of htra-2 alpha cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2 alpha isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2 alpha to regulate dsx is female-specific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2 alpha has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.

A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA

Female-specific splicing of Drosophila doublesex (dsx) pre-mRNA is regulated by the products of the transformer (tra) and transformer 2 (tra2) genes. In this paper we show that Tra and Tra2 act by recruiting 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 general splicing factors are sufficient to commit dsx pre-mRNA to female-specific splicing, and individual SR proteins differ significantly in their ability to participate in commitment complex formation. Characterization of the proteins associated with affinity-purified complex formed on dsx pre-mRNA reveals the presence of Tra, Tra2, SR proteins, and additional unidentified components. We conclude that Tra, Tra2, and SR proteins are essential components of a splicing enhancer complex.

Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro

Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing. Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins. The mechanism by which these proteins regulate RNA processing has not been characterized. In this paper we show that tra-2 produced in Eacherichia coll binds specifically to a site within the female-specific exon of dsx pre-mRNA. This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation. These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing.