Project description:The quality control and export of mRNA by RNA-binding proteins are necessary for the survival of malaria parasites, which have complex life cycles. Malarial nuclear poly(A) binding protein 2 (NAB2), ALWAYS EARLY (ALY) and serine/arginine-rich (SR) proteins, such as nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and SR1, are involved in nuclear mRNA export in malaria parasites. However, their functions and cellular localization are not fully understood. In this study, we found that NAB2 and SR1, but not ALY, NPL3 or GBP2, played essential roles in the asexual development of malaria parasites, while GBP2 was involved in gametocyte production. Moreover, GBP2 localized to both the nucleus and cytoplasm of malaria parasites and interacted with the proteins ALBA4, DOZI and CITH, which play roles in translational repression. Our findings suggest that malarial GBP2 may be involved in the recruitment of ALBA4, DOZI and CITH. Immunoprecipitation coupled to mass spectrometry (IP-MS) revealed that PHAX domain-containing protein, an adaptor protein for exportin-1, also interacted with GBP2, suggesting that mRNA export occurs via the PHAX domain-containing protein pathway in malaria parasites. Fluorescence live cell imaging revealed that malarial NAB2 localized at the nuclear periphery and co-localized with NUP205. Moreover, using IP-MS, we found that malarial NAB2 interacted with transportin. RNA immunoprecipitation coupled to RNA sequencing revealed that malarial NAB2 bound directly to 143 mRNAs, including those encoding 40S and 60S ribosomal proteins. This indicates that malarial NAB2 is involved in general mRNA assembly and is shuttled between the nucleus and cytoplasm. Our findings suggest that unique mRNA export and post-transcriptional gene regulation mediated by RNA-binding proteins occur in malaria parasites.
Project description:Transcription profiling was performed on the time course of Nab2 protein depletion in THC-Nab2 strain at 0h, 7h, 12h in biological triplicates to access its affect on the composition and structure of mRNA in budding yeast. For the protein depletion experiment THC-NAB2 (THJ collection Y2484, THC-NAB2:KAN tTA:URA, Open Biosystems) strain was used, where expression of the NAB2 gene is under control of a tetracyclin-repressible promoter.
Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3M-bM-^@M-^Y-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of a conditional degron nab2 mutant that allows the depletion of the protein in 15 minutes, to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation. Two and three repeats of the wild type control and the nab2-td mutant respectively at time 0, 30 and 75 minutes after the degron induction.
Project description:The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver, using DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate (FDR) into account, we extracted the top 1,000 genes that were expressed differentially between fed and fasted rats. In all three tissues, Gene Ontology analysis revealed marked changes in the expression of âmetabolismâ category genes and a hypergeometric test demonstrated that within this category, lipid and protein biosynthesis-related genes were down-regulated. These findings indicate simultaneous down-regulation of genes involved in energy-consuming pathways in the BAT, WAT and liver of fasted rats. In the BAT of fasted rats, there was marked up-regulation of genes in the âprotein ubiquitinationâ category, suggesting that the ubiquitin-proteasome system is involved in saving energy as an adaptation to food shortage. Experiment Overall Design: Rats (7-week-old) were given a commercial diet between 10:00 and 16:00 for 7 days. At 10:00 on day 8, they were divided into two groups that comprised animals of similar body weights. One group continued to be fed as described above (fed group, n=4 for array analysis), whereas the other group were not fed (fasted group, n=4 for array analysis). Both groups received water ad libitum. At 16:00 on day 8, the liver, interscapular BAT and perinephrial WAT were excised, and analyzed for fasting effect.
Project description:We exploit the predictable time course of Drosophila brain development to perform a temporally coupled quantitative proteomic analysis of the pupal brain in Nab2 mutant or overexpression models, which reveals that Nab2 is required to regulate the abundance of a number of proteins with critical roles in Drosophila neurons. Pupal brains lacking Nab2 show dysregulation of proteins, such as Futsch, Turtle, Contactin, and Van Gogh, that typically function in brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycle, and other neurodevelopmental processes. Overall, these data define a role for Nab2 during neurodevelopment in regulating protein abundance for a subset of the brain proteome and provide a window into the potential functions of human ZC3H14 protein.
Project description:Previously, we constructed a coculture model to analyze the effect of macrophages on intestinal epithelial cells, and found that TNF-a secreted from human macrophage-like THP-1 cells induced cell damage to intestinal epithelial Caco-2 cells (Exp.Cell.Res. 2006, 312(19):3909-19). In this study, we present activation of NF-kB in Caco-2 cells within 15 min after coculturing. To reveal how TNF-a secreted from THP-1 cells affects Caco-2 cells in an early stage of coculture, we exhaustively analyzed the changes of gene expression in Caco-2 cells cocultured with THP-1 cells over the time periods of 0, 1, 3, 6, 24, and 48 h by using a DNA microarray. Differentially expressed genes extracted with maSigPro demonstrated that IEX-1 was the lowest p-value gene, that is, the most significantly changed gene among the up-regulated genes. The genes expressed in a similar pattern to IEX-1 involved immunity, apoptosis, and protein kinase cascade. These findings suggest that the stimuli of TNF-a from THP-1 cells activates NF-kB, leading induction of various gene expression. This pattern of gene expression indicates that not only early defense response but also cell death occurs at the same time, causing inflammatory condition. Caco-2 cells were cultured for 14 days on a semi-permeable support, and THP-1 cells were differentiated with phorbol myristate acetate (PMA) for 4 days in 12-well plates. Then, the semi-permeable support membrane in which Caco-2 cells had been cultured was placed on the 12-well plates on which THP-1 has been cultured.
Project description:The effects of the administration of molecular hydrogen-saturated drinking water (hydrogen water) on hepatic gene expression were investigated in rats. Using DNA microarrays, 548 upregulated and 695 downregulated genes were detected in the liver after a 4-week administration of hydrogen water. Gene Ontology analysis revealed that genes for oxidoreduction-related proteins, including hydroxymethylglutaryl CoA reductase, were significantly enriched in the upregulated genes. 4-week-old male Sprague-Dawley rats were acclimatized for 4 days in a room maintained at 24 ± 3°C with a relative humidity of 55 ± 15% with a 12-h light-dark cycle (lights on at 7:00, off at 19:00) and were fed ad libitum on a commercial diet (MF, Oriental Yeast Co., Yokohama, Japan). After acclimatization, the rats were divided into two groups (n = 8 per group) with similar mean body weights. During a period of 4 weeks, one group (control group) was supplied with sterilized distilled water and the other (HW group) with hydrogen water produced by MiZ Co. Ltd. (Fujisawa, Japan), which contains 0.7 mM dissolved hydrogen (pH 7.4). All the animals drank water ad libitum. After 4 weeks, each rat was anesthetized with diethyl ether and blood samples were collected from the abdominal aorta. The liver was then excised and analyzed the effect of hydrogen water extract administration on hepatic gene expression.
Project description:We have previously showed that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than an identical composition of amino acids mixture does. The present study was conducted to investigate a comparative effect of WPH on gene expression. Male Sprague-Dawley rats subjected to a 2-h swimming exercise were administered either a carbohydrate-amino acid diet or a carbohydrate-WPH diet immediately after exercise. One hour after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. As a result, ingestion of WPH altered 189 genes in considering the false discovery rate. Among the upregulated genes, 8 Gene Ontology (GO) terms were enriched, which included key elements in muscle repair after exercise such as Cd24, Ccl2, Ccl7 and Cxcl1. On the other hand, 9 GO terms were enriched in the gene sets downregulated by ingestion of WPH and these GO terms fell into 2 clusters, 'regulation of ATPase activity' and 'immune response'. Furthermore, we found that WPH activate the 2 upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1a (HIF-1a), which may act as key factors for regulation of gene expression. These results suggest that ingestion of WPH, compared to an identical composition of amino acid mixture, induces greater changes in the after-exercise gene expression profile via activation of the proteins, ERK1/2 and HIF-1a. Male Sprague-Dawley rats (5-week-old) with body weights of approximately 150 g each (CLEA Japan, Inc., Tokyo, Japan) were used in this study. Rats were maintained at 23 +/- 2C, with lights on from 8:00 to 20:00 and off from 20:00 to 8:00. Rats had free access to food (protein 23.6%, fat 5.3%, carbohydrate 54.4%, ash 6.1%, fiber 2.9%, moisture 7.7%, MF, Oriental Yeast Co., Ltd., Osaka, Japan) and water. After 2- or 3-day acclimation, the rats were pre-trained to swimming exercise for 3 days. One day before the experiment, they were fed 5 g of a restricted diet (MF, Oriental Yeast Co., Ltd., Osaka, Japan). On the day of the experiment, rats swam for 2 hours, with 4 rats swimming simultaneously in a barrel filled with water to a depth of 50 cm, allowing an average surface area of 400 cm2 for each animal. The water temperature was maintained at a constant of 35C. Immediately following exercise, rats were given one of two isoenergetic test solutions by gavage. These solutions contained 44 kJ in a four-test dose that represented about 15% of daily energy needs and included either a mixed meal containing carbohydrate and amino acid mixture (AAM) or a mixed meal containing carbohydrate and whey protein hydrolysate (WPH; Meiji Co., Ltd., Japan). This study was approved by the Animal Committee of Food Science Research Lab., Meiji Co., Ltd., with the animals receiving care according to the guidelines laid down by this committee.
Project description:Persimmon (Diospyros kaki L. f.) is a most popular fruit in Asian countries but its peels are totally wasted despite of containing a plenty of antioxidants such as carotenoids and polyphenols. We prepared a fat-soluble extract from a persimmon peel (PP) fraction and fed type 2 diabetic Goto-Kakizaki (GK) rats with a PP extract-containing AIN-93G diet (PP diet) for 12 weeks. Compared with the control AIN-93G diet, the feeding of the PP diet reduced the plasma glutamic-pyruvate transaminase activity significantly, with accumulation of M-NM-2-cryptoxanthin in the liver. A DNA microarray analysis revealed that the PP diet altered the hepatic gene expression profiles. In particular, insulin signaling pathway-related genes were significantly enriched in differentially expressed gene sets. Moreover, Western blotting analysis actually showed the promotion of IRM-NM-2 tyrosine phosphorylation. All these data suggest that the PP extract administration to the GK rats improves their insulin resistance. Male GK rats (90M-bM-^@M-^S107 g), aged 5 weeks, were purchased from Japan SLC Co. (Hamamatsu, Japan). The rats were maintained in a room at 22 M-BM-1 1M-BM-0C under a 12-h light-dark cycle (lights on at 0800) and administered a commercial diet (AIN-93G, Research Diets, Inc., New Brunswick, NJ, USA) for a week. The rats were allowed free access to diet and water. After one week, they were divided into two groups with similar average body weights. The control group rats (n = 4) were fed on the commercial diet, and the PP group rats (n = 4) on the same diet containing the PP extract. After the 12-week-feeding the liver was excised and analyzed the effect of PP extract administration on hepatic gene expression.