The Germ Cell Nuclear Proteins hnRNP G-T and RBMY Activate a Testis-Specific Exon

The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2β is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5′ splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2β and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3′ splice site normally weakly selected in the testis. Co-expression of Tra2β with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein

A Novel Dietary Source of EPA and DHA: Metabolic Engineering of an Important Freshwater SpeciesCommon Carp by fat1-Transgenesis

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are essential for neural development and human health. The n-3 PUFAs are mainly obtained from marine fish by dietary intake. Freshwater fish species usually contain low level of n-3 PUFAs due to the lack of n-3 PUFAs in their food chain. In this study, we report on the substantial production of EPA and DHA in a globally important freshwater fish species, common carp (Cyprinus carpio). This was achieved by introducing an all-fish transgene CA:fat1 containing the fish codon-optimized omega-3 desaturase gene (fat1) driven by the common carp -actin promoter (CA). Through a sperm sample screening method, we successfully generated fat1-positive F1 transgenic population with high efficiency. In F1 population, the muscle contents of ALA (18:3n-3), EPA and DHA were significantly increased when compared with non-transgenic siblings. Thereafter, four independent F2 heterozygous lines were obtained from 4 F1 transgenic males and a detailed comparison of fatty acids profile and growth performance was carried out for these 4 lines. All fat1-transgenic common carps from 4 lines showed an evident decrease in n-6 PUFA contents and a substantial increase in n-3 PUFA contents, among which line 4 stands out, showing a statistically significant increase in all 4 types of n-3 PUFAs including ALA (4.4-fold increase, p<0.001), EPA (4.8-fold increase, p<0.01), C22:5n-3 (DPA, 2.4-fold increase, p<0.05), and DHA (1.9-fold increase, p<0.05). Therefore, the line 4 was selected as the optimized breeding stock for further study, and the proximate nutrition composition and PUFA synthesis pathway were analyzed. Our study demonstrates that in the transgenic group, the muscular lipid content did not change, while fat accumulations in the internal organs and especially in the liver were significantly decreased as a result of hyperactivation of fatty acid oxidation process. Finally, we conclude that the all-fish CA:fat1-transgenic freshwater fishcommon carpcan serve as a novel healthy dietary source of n3-PUFAs, especially EPA and DHA

Evaluating estrogenic and anti-estrogenic effect of endocrine disrupting chemicals (EDCs) by zebrafish (Danio rerio) embryo-based vitellogenin 1 (vtgl) mRNA expression

By measuring the vitellogenin 1 (vtgl) expression through quantitative PCR and in situ hybridization, we used the zebrafish embryo as an in vivo model to access the estrogenic or anti-estrogenic effects of several endocrine disrupting chemicals (EDCs), such as natural estrogen 17 beta-estradiol (E2), estriol (E3), synthetic hormones including diethylstilbestrol (DES), 4-octyl phenol (OP), bisphenol A (BPA), tamoxifen (TMX) and 3-(2,3-dibromopropyl) isocyanurate (TBC). According to our data, the estrogenic effect Of the tested chemicals was ranked as: DES &gt; E-2 &gt; E-3 &gt; OP &gt; BPA, which is consistent with various in vivo and in vitro models. Therefore, the measurement of vtgl gene expression in zebrafish embryos would be a valuable method for screening EDCs including both environmental estrogens and anti-estrogens