YB-1 Binds to GluR2 mRNA and CaM1 mRNA in the Brain and Regulates their Translational Levels in an Activity-Dependent Manner

The translational regulator YB-1 binds to mRNAs. In the brain, YB-1 is prominently expressed from the prenatal stage until the first week after birth, being associated with polysomes and distributed in neuronal dendrites, but its expression declines to a much lower level thereafter. It is therefore of interest to identify the mRNAs whose translation is controlled by YB-1 in the postnatal growing brain. In this study we found that YB-1 interacted with the mRNAs for glutamate receptor subunit 2 (GluR2) and calmodulin1 (CaM1) in both brain and NG108-15 cells. Overexpression or knockdown of YB-1 altered the levels of these proteins significantly in cultured cells without any change in their mRNA levels. When the cells were treated with neurotransmitters, translation of these proteins was induced within a short time, and a change in the amount of YB-1 on its target mRNAs was observed in the heavy-sedimenting polysome fractions on a sucrose gradient. Depletion of YB-1 expression by siRNA abrogated the translational activation. Furthermore, in the brain of kainic acid-treated mice, the distribution of YB-1 was shifted to much heavier fractions associated with polysomes within 30 min to 1 h after the treatment, and the distribution returned to lighter fractions within the following 2 h. The protein levels of GluR2 and CaM1 were also increased transiently when the distribution of YB-1 on the gradient changed. These results suggest that in the brain of growing mice, YB-1 binds to GluR2 and CaM1 mRNAs and regulates their translation in an activity-dependent manner.     

Roles of YB-1 under arsenite-induced stress: Translational activation of HSP70 mRNA and control of the number of stress granules

Background

When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.

Method

Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.

Results

Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.

Conclusion

In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.

General significance

Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.     

HSP60 interacts with YB-1 and affects its polysome association and subcellular localization

YB-1 is a DNA/RNA-binding protein which, in the cytoplasm, associates with polysomes and regulates translation. However, YB-1 has a novel nuclear localization signal, and its nuclear accumulation is correlated with cancer induction. Here we designated the amino-acid sequence as YB-NLS and demonstrated that YB-NLS is necessary for the nuclear translocation of overexpressed YB-1 in NG108-15 cells. In addition, we found that a heat shock protein, HSP60, binds to YB-NLS in the cytoplasm. Interestingly, when HSP60 expression was repressed, an increase of polysome-associated YB-1 was observed in heavy-sedimenting fractions on a sucrose gradient. Overexpression of HSP60 resulted in a decrease of YB-1 in the heavy-sedimenting fractions and suppression of YB-NLS activity. Furthermore, the NLS-deleted YB-1 was apparently associated with the heavy-sedimenting polysomes. These results suggest that HSP60 interacts with YB-1 at the YB-NLS region and acts as a regulator of polysome association and the subcellular distribution of YB-1.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.     

An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1

Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.     

Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

Polysomes, Messenger RNA, and Growth in Soybean Stems during Development and Water Deficit

The polysome status and populations of polysomal mRNA were examined in different regions of dark-grown soybean (Glycine max [L.] Merr.) stems that contained either dividing, elongating, or mature (nongrowing) cells. There was a developmental gradient of polysome content in which the dividing tissue had the highest levels and the mature tissue the lowest. A few hours after transplanting the seedlings to vermiculite having low water content (water potential Ψ_w = −0.29 megapascals), stem growth rate decreased to 30% of well-watered controls and the polysome content decreased most in the dividing and elongating tissues. After 24 to 36 hours, stem growth and polysome content recovered gradually. In vitro translation products of polysomal mRNA from dividing, elongating or mature tissue were examined on two-dimensional gels. In well-watered controls, each of the stem regions was enriched in a small subset of the polysomal mRNA population, probably because of developmentally regulated gene expression. Exposing plants to low Ψ_w for 24 hours induced a change in the relative abundance of a small number of polysomal mRNAs in the elongating and mature tissues, but not in the dividing tissue. After 24 to 72 hours at low Ψ_w, the changes in polysomal mRNA population were reversed in the elongating tissue. The data indicate that changes in stem growth at low water potential are associated with changes in polysome status and polysomal mRNA in the elongating tissue.     

银鲫果糖-1,6-二磷酸酶的分子克隆与表达分析

果糖-1,6-二磷酸酶(EC 3.1.3.11)是糖异生中的关键限速酶之一, 在糖代谢中起重要作用。哺乳动物存在肝脏型和肌肉型两种果糖-1,6-二磷酸酶同工酶,分别由Fbp1和Fbp2编码。银鲫作为我国重要的经济养殖鱼类, 尚无果糖-1,6-二磷酸酶基因的有关资料, 其组织分布特征和胚胎发育模式亦不清楚。本研究采用RACE方法从银鲫原肠胚SMART cDNA文库中扩增了果糖-1,6-二磷酸酶基因的全长cDNA, 其长度为1170 bp,编码337个氨基酸残基,多重序列比对和系统发育分析表明该基因为肝脏型果糖-1,6-二磷酶。RT-PCR分析虽在银鲫的肝、脑、心、脾、肾、肠、肌肉和卵巢组织中皆能检测到该基因的表达, 但以肝组织的表达量最高。Western Blot检测表明, 肝脏组织除有一条与其他组织(肌肉除外)共有的蛋白带之外,还有一条特异带;肌肉中有不同于其他组织的特异带。成熟卵子和不同发育阶段胚胎的RT-PCR和Western Blot分析都可检测到母源的CagFbp转录本和蛋白,且其转录本从原肠期开始上升, 到神经胚时迅速上升到较高水平, 其蛋白从尾芽期以后出现一条比母源蛋白分子量小、与肝脏的特异带大小基本相同的蛋白带。这些结果证实本研究克隆的CagFbp为肝脏型,且鱼类至少存在肝脏型和肌肉型两种果糖-1,6-二磷酸酶同工酶。       

Tissue-specific expression of c-series gangliosides in the extraneural system

c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner.