Transformer-2 Protein Research Articles

MicroRNA-330-3p represses the proliferation and invasion of laryngeal squamous cell carcinoma through downregulation of Tra2β-mediated Akt signaling

MicroRNAs (miRNAs), a type of post-transcriptional regulators, exert a crucial role in the malignant progression of laryngeal squamous cell carcinoma (LSCC). MicroRNA-330-3p (miR-330-3p), a recently identified tumor-associated miRNA, is implicated in multiple cancers. Yet, the relevance of miR-330-3p in LSCC remains unexplored. The findings of our study demonstrated a lower expression of miR-330-3p in LSCC. Functional assays revealed that upregulation of miR-330-3p marked restricted the proliferation, colony formation and invasion of LSCC cells. Transformer-2 protein homolog beta (Tra2β) was identified as a target gene of miR-330-3p. An inverse correlation between miR-330-3p and Tra2β mRNA expression was evidenced in LSCC specimens. The upregulation of miR-330-3p significantly repressed Tra2β expression and the phosphorylation of the Akt protein. In addition, Tra2β overexpression markedly abrogated the tumor suppressive role of miR-330-3p in LSCC cells. Overall, our results uncovered that miR-330-3p exerted a tumor-inhibition function in LSCC through targeting Tra2β to inhibit Akt activation.

Hepatic proteomic analysis of selenoprotein F knockout mice by iTRAQ: An implication for the roles of selenoprotein F in metabolism and diseases

Selenoprotein F (Selenof) is an endoplasmic reticulum (ER)-resident protein. It may be functionally linked to glycoprotein folding in the ER but the detail function is not fully understood. To study the function of Selenof, we used CRISPR/Cas9 to generate Selenof knockout mice and performed proteomic analysis of hepatic proteins by iTRAQ. Collectively, 83 differently expressed proteins (DEPs) were identified in the liver of Selenof knockout mice. The changes of mRNA and protein levels of 6 selected DEPs, Fatty acid synthase, ATP-citrate synthase, Glutathione S-transferase P 1, Transformer-2 protein homolog beta, Pyruvate kinase, Metallothionein-2, were further verified by quantitative real-time PCR or Western blot. The roles of 83 DEPs are mainly related to metabolism and cancer. Consistently, the levels of NADPH and ATP, two molecules closely related to energy metabolism, are significantly changed in the livers of Selenof knockout mice. SignificanceOur study identified potential biological pathways and proteins related to Selenof deletion. These findings will provide possible proteins/pathways related to Selenof and help to understand the function of Selenof as well as the relationship between Selenof and certain diseases.

Tra2β silencing suppresses cell proliferation in laryngeal squamous cell carcinoma via inhibiting PI3K/AKT signaling.

Transformer-2 protein homolog beta (Tra2β) generally plays an important role in various human cancers, but its role and the underlying mechanisms in laryngeal squamous cell carcinoma (LSCC) remained unknown. So this study aimed to assess the clinical significance and regulatory mechanisms of Tra2β in LSCC. Laboratory analysis. Expression of Tra2β was compared in human LSCC tissue samples and paired adjacent normal tissue samples. The in vitro effects of Tra2β expression in Hep-2 cells on their proliferation, invasion, and migration were assessed by CCK-8 assays, Matrigel invasion, and transwell migration assays. In addition, the effects of downregulation of Tra2β on the activation of PI3K/AKT signaling pathway were measured using Western blot analysis. The effect of Tra2β on the growth of tumors was detected in the Hep-2-injected xenograft models in vivo. Reverse-transcription quantitative polymerase chain reaction analysis and immunochemistry analysis indicated that the increased expression of Tra2β in LSCC was significantly associated with poor differentiation, lymph node metastasis, and advanced clinical stage. In vitro knockdown of Tra2β caused a significant decrease in the proliferation, invasion, and migration of Hep-2 cells. Tra2β silencing decreased the expression of Bcl-2 but increased Bax and Caspase-3 both in mRNA and protein levels. Furthermore, knockdown of Tra2β eliminated the suppressive effects of activation of PI3K/AKT signaling. In vivo knockdown of Tra2β significantly inhibited the tumor growth of Hep-2-injected xenograft mice. The results of the present study demonstrated that knockdown of Tra2β inhibits the proliferation and invasion of LSCC cells, at least partly via inhibiting PI3K/AKT signaling. NA Laryngoscope, 129:E318-E328, 2019.

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.

The gene transformer-2 of Sciara (Diptera, Nematocera) and its effect on Drosophila sexual development.

BackgroundThe gene transformer-2, which is involved in sex determination, has been studied in Drosophila, Musca, Ceratitis, Anastrepha and Lucilia. All these members of Diptera belong to the suborder Brachycera. In this work, it is reported the isolation and characterisation of genes transformer-2 of the dipterans Sciara ocellaris and Bradysia coprophila (formerly Sciara coprophila), which belong to the much less extensively analysed Sciaridae Family of the Suborder Nematocera, which is paraphyletic with respect to Suborder Brachycera.ResultsThe transformer-2 genes of the studied Sciara species were found to be transcribed in both sexes during development and adult life, in both the soma and germ lines. They produced a single primary transcript, which follows the same alternative splicing in both sexes, giving rise to different mRNAs isoforms. In S. ocellaris the most abundant mRNA isoform encoded a full-length protein of 251 amino acids, while that of B. coprophila encoded a protein of 246 amino acids. Both showed the features of the SR protein family. The less significant mRNA isoforms of both species encoded truncated, presumably non-functional Transformer-2 proteins. The comparison of the functional Sciara Transformer-2 proteins among themselves and those of other insects revealed the greatest degree of conservation in the RRM domain and linker region. In contrast, the RS1 and RS2 domains showed extensive variation with respect to their number of amino acids and their arginine-serine (RS) dipeptide content. The expression of S. ocellaris Transformer-2 protein in Drosophila XX pseudomales lacking the endogenous transformer-2 function caused their partial feminisation.ConclusionsThe transformer-2 genes of both Sciaridae species encode a single protein in both sexes that shares the characteristics of the Transformer-2 proteins of other insects. These proteins showed conserved sex-determination function in Drosophila; i.e., they were able to form a complex with the endogenous Drosophila Transformer protein that controls the female-specific splicing of the Drosophila doublesex pre-mRNA. However, it appears that the complex formed between the Drosophila Transformer protein and the Sciara Transformer-2 protein is less effective at inducing the female-specific splicing of the endogenous Drosophila doublesex pre-mRNA than the DrosophilaTransformer-Transformer2 complex. This suggests the existence of species-specific co-evolution of the Transformer and Transformer-2 proteins.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects.

BackgroundIn the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2.ResultsThe gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features.ConclusionsThese results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-β

RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.

Isolation, Sequence Identification and Tissue Expression Distribution of Three Novel Porcine Genes-UBC1, TRA2A and SH21A

Liu G.Y., Zhao, S.M., Song, X.L., Wang, J. and Gao, S.Z. 2008. Isolation, sequence identification and tissue expression distribution of three novel porcine genes-UBC1, TRA2A and SH21A. J. Appl. Anim. Res., 33:33–38. Ubiquitin-conjugating enzyme E2–25 kDa (UBC1), transformer-2 protein homolog TRA-2 alpha (TRA2A) and SH2 domain protein 1A (SH21A) genes are most important genes highly related or potentially related with the porcine production. The complete coding sequences of these three porcine genes were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referred highly homologous pig ESTs. Porcine UBC1 gene encodes a protein of 200 amino acids which contains the conserved putative UBCc domain and has high homology with the UBC1 proteins of three species-human, mouse and bovine (100%). The porcine TRA2A gene encodes a protein of 279 amino acids that contains the conserved putative RNA recognition motif (RRM) domain and has high homology with the TRA2A proteins of two species-human (99%) and mouse (97%). The porcine SH21A gene encodes a protein of 128 amino acids that contains the conserved putative src homology 2 (SH2) domain and has high homology with the SH21A proteins of two species human (92%) and mouse (86%). The tissue expression analysis indicated that the swine UBC1, TRA2A and SH21A genes were expressed in different tissues including lung, fat, small intestine, large intestine, liver, muscle, spleen and kidney. Our experiment is the first to establish the primary foundation for further research on these three swine genes.

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.

Binding of the Drosophila transformer and transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing.

Sex-specific alternative processing of double-sex (dsx) precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, we identified six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer (tra) and transformer-2 (tra-2) products bind to the 13-nucleotide sequences of dsx pre-mRNA. These results strongly suggest that the female-specific splicing of dsx pre-mRNA is activated by binding of these proteins to the 13-nucleotide sequences.