The Proteins Interacting with Prmt5 in Medaka (Oryzias latipes) Identified by Yeast Two-Hybridization.

BACKGROUND: Prmt5 plays major role in regulation of gene expression, RNA processing, cell growth and differentiation, signal transduction, germ cell development, etc., in mammals. Prmt5 is also related to cancer. Knowing the proteins interacting with Prmt5 is important to understand Prmt5's function in cells. Although there have been reports on proteins binding with Prmt5 in mammals, the partner proteins of Prmt5 in fish are still unclear. OBJECTIVES: The objective was to obtain proteins that bind with Prmt5 in medaka, a model fish. METHODS: Yeast two hybridization was adopted to achieve the objective. Medaka Prmt5 was used as a bait to fish the prey, binding proteins in a cDNA library of medaka. Co-immunoprecipitation and in silicon analysis were performed to study the interaction of medaka Mep50 and Prmt5. RESULTS: Eight proteins were identified to bind with Prmt5 from 69 preliminary positive colonies. The binding proteins are methylosome protein 50 (Mep50), apolipoprotein A-I-like (Apo-AI), PR domain containing protein 1a with zinc fingers (Prdm1a), Prdm1b, T-cell immunoglobulin mucin family member 3 (Tim-3), phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (Paics), NADH dehydrogenase subunit 4 (ND4) and sciellin (Scl). Co-immunoprecipitation confirmed the interaction of medaka Prmt5 and Mep50. Predicted structures of medaka Prtm5 and Mep50 are similar to that of human PRMT5 and MEP50. CONCLUSION: Medaka Mep50, Prdm1a, Prdm1b, Apo-AI, Tim-3, Paics, ND4, and Scl bind with Prmt5.

Identification, expression pattern, and immune response of Tim-1 and Tim-4 in embryos and adult medaka (Oryzias latipes).

T-cell immunoglobulin (Ig) and mucin domain-containing 1 (Tim-1) and Tim-4 are two members of the Tim family. In mammals, Tim-1 and Tim-4 are proteins mainly expressed in immune cells and are associated with immune response. In the present study, medaka Oryzias latipes' Tim-1 (OlTim-1) and OlTim-4 were identified and characterized using bioinformatics analyses. With the use of reverse-transcription polymerase chain reaction, the expression profiles of OlTim-1 and OlTim-4 were examined in embryos and adult fish and in immune tissues following the intraperitoneal injection of stimulants. The results revealed that OlTim-1 possesses a cytoplasmic region, a transmembrane region, a mucin domain, and an Ig-like domain, while OlTim-4 is composed of two Ig-like domains and a mucin domain, but without the transmembrane region and cytoplasmic region. OlTim-1 and OlTim-4 expressions are detectable from the gastrula stage on, indicating that they are zygotic genes. Furthermore, OlTim-1 and OlTim-4 are expressed ubiquitously in the adult. Administration of immune stimulants, namely lipopolysaccharides and polyinosinic:polycytidylic acid, significantly increased the expression levels of OlTim-1 and OlTim-4 in the liver and intestine within 1 day and in the head, kidney, and spleen within 3 to 4 days postinjection. These results suggest that OlTim-1 and OlTim-4 are possibly involved in both innate and adaptive immunities.

Fundc1 is necessary for proper body axis formation during embryogenesis in zebrafish.

FUN14 domain-containing protein 1 (FUNDC1) is a mitochondrial outer membrane protein which is responsible for hypoxia-induced mitophagy in mammalian cells. Knockdown of fundc1 is known to cause severe defects in the body axis of a rare minnow. To understand the role of Fundc1 in embryogenesis, we used zebrafish in this study. We used bioimaging to locate zebrafish Fundc1 (DrFundc1) with MitoTracker, a marker of mitochondria, and/or CellLight Lysosomes-GFP, a label of lysosomes, in the transfected ovary cells of grass carp. The use of Western blotting detected DrFundc1 as a component of mitochondrial proteins with endogenous COX IV, LC3B, and FUNDC1 in transgenic human embryonic kidney 293 T cells. DrFundc1 induced LC3B activation. The ectopic expression of Drfundc1 caused cell death and apoptosis as well as impairing cell proliferation in the 293 T cell line, as detected by Trypan blue, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and incorporation of BrdU. DrFundc1 up-regulated expression of both autophagy- and apoptosis-related genes, including ATG5, ATG7, LC3B, BECLIN1, and BAX in transgenic 293 T cells. A knockdown of Drfundc1 using short hairpin RNA (shRNA) led to midline bifurcation with two notochords and two spinal cords in zebrafish embryos. Co-injection of Drfundc1 mRNA repaired defects resulting from shRNA. Knockdown of Drfundc1 resulted in up- or down-regulation of genes related to autophagy and apoptosis, as well as decreased expression of neural genes such as cyclinD1, pax2a, opl, and neuroD1. In summary, DrFundc1 is a mitochondrial protein which is involved in mitophagy and is critical for typical body axis development in zebrafish.

Bcl6aa and bcl6ab are ubiquitously expressed and are inducible by lipopolysaccharide and polyI:C in adult tissues of medaka Oryzias latipes.

B-cell lymphoma-6 (Bcl6) is a transcriptional repressor that plays important roles in various physiological activities such as innate and adaptive immune response, lymphocyte differentiation, and cell cycle regulation in mammals. Two homologs of Bcl6a, namely Bcl6aa and Bcl6ab, are identified in teleost fish including medaka Oryzias latipes. The expression profiles of bcl6aa and bcl6ab in medaka were studied using reverse-transcription polymerase chain reaction and in situ hybridization. The transcripts of bcl6aa and bcl6ab were detected from very early embryos such as the four-cell stage until hatching. Bcl6aa and bcl6ab were clearly detected in the embryonic body from 5 days postfertilization onward by in situ hybridization. Bcl6aa was specifically expressed in the retina, whereas bcl6ab was expressed in entire embryonic body. The results referred to that both bcl6aa and bcl6ab originate maternally in the zygotes and may play major roles in embryogenesis of medaka. The transcripts of bcl6aa and bcl6ab were detected in all examined adult tissues, including immune organs such as the gill, spleen, kidney, liver, and intestine. The expression of bcl6aa and bcl6ab in the liver, spleen, head-kidney, and intestine could be upregulated or downregulated by lipopolysaccharide and polyriboinosinic-polyribocytidylic acid. These results indicate that both bcl6aa and bcl6ab may be involved in immune response in medaka.

Identification, characterization, expression profiles of OlHavcr2 in medaka (Oryzias latipes).

Hepatitis A virus cellular receptor2 (Havcr2) also named T-cell immunoglobulin and mucin domain containing-3 (Tim-3) was initially described as a T helper 1-specific cell surface protein, a member of Tim family implicated in the regulating process of adaptive and innate immune responses. Here, medaka (Oryzias latipes) Havcr2 (OlHavcr2) was isolated and characterized. Unlike other Havcr2 proteins, OlHavcr2 possesses two Ig-like domains but lacks cytoplasmic and transmembrane domains. RT-PCR results revealed that OlHavcr2 mRNA was expressed strongly in the liver, moderately in the intestine, heart and ovary, and weakly in the muscle, gill, brain, eye, spleen, and testis. OlHavcr2 expression begun from gastrula stage and was maintained until hatching. The signal of OlHavcr2 was mainly identified in the blood system in the yolk sac by in situ hybridization. These results indicated that OlHavcr2 is expressed ubiquitously in adult tissues, and is a zygotic gene expressed from gastrula onwards in embryogenesis. OlHavcr2 may play a significant role in the blood system of medaka. In the immune organs, OlHavcr2 expression was affected by the immune stimulants, lipopolysaccharide and poly I:C, suggesting that OlHavcr2 was involved in innate immunity and adaptive immunity in medaka.

Expression pattern and functional analysis of fundc1 in rare minnow (Gobiocypris rarus).

Fundc1 is a mitochondrial outer membrane protein and plays important roles in mitochondria fission and hypoxia-induced mitophagy in mammalian cells. However, there is no relevant report of fundc1 in fish. In the present study, we cloned a 942bp fundc1 cDNA from rare minnow. The cDNA, designated as Grfundc1 cDNA, contains an open reading frame (ORF) of 459bp which encodes a polypeptide of 152 amino acid residues. Comparisons of deduced amino acid sequences demonstrated that Grfundc1 was highly homologous with those of other vertebrates. RT-PCR and real time PCR detection revealed that the transcripts of Grfundc1 were not detectable in the unfertilized eggs and had high levels at blastula and gastrula stages. Whole mount in situ hybridization analysis observed that Grfundc1 was ubiquitously expressed at early stage and later riched in specific regions, such as brain, branchial arch, eye and somite during embryogenesis. Grfundc1 was expressed in all the tissues of rare minnow adult, including brain, liver, gill, eyes, heart, kidney, intestine, muscle, testis and ovary. The expression of Grfundc1 in the brain, gill, heart and eye of rare minnow adult was significantly down-regulated by hypoxia. Similar hypoxic response was observed in the rare minnow embryos at 48hpf following hypoxia exposure. Functional analysis showed that knockdown of Grfundc1 significantly caused defects in the body axis and dorsal neural tissues of rare minnow embryos. These results indicate that Grfundc1 may play important roles in embryogenesis in fish.