Sachiyo Ohashi

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.

Isolation and characterization of brain Y-box protein: developmentally regulated expression, polyribosomal association and dendritic localization.

Y-box proteins are DNA- and RNA-binding proteins and control specific gene expression at both transcriptional and translational levels. Particularly in germ cells, it has been reported that Y-box proteins bind to paternal or maternal mRNAs to form mRNPs, mask them from translation and control cell maturation. In this study, we cloned cDNA for a Y-box protein from rat brain. A deduced amino acid sequence of the protein was very similar to that of several other Y-box proteins, and we termed the protein rBYB1 (rat brain Y-box protein 1). rBYB1 was found to be considerably expressed in the cytoplasm of pre- and early postnatal brains, and then decreased to adult levels with brain development. Further, we found rBYB1 to be distributed in both polyribosomal and nonpolyribosomal (mRNP) fractions on a sucrose density gradient, and to be associated with polyribosomes via RNA in the higher-density fractions. Moreover, rBYB1 was localized in dendrites of the primary hippocampal neurons. We compared these sucrose gradient and intracellular rBYB1 localization results with those for fragile X mental retardation protein (FMRP), which is known to be an mRNA-binding and polyribosome-associating translational regulator distributed in neuronal dendrites. Our results suggest that in the brain of prenatal and newborn animals, rBYB1 may function in storage and/or translational regulation of mRNAs involved in the rapid progress of the postnatal brain, and in mature neurons, it may also participate in the control of protein synthesis in dendrites.

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.

Mechanism of YB-1-mediated translational induction of GluR2 mRNA in response to neural activity through nAChR

BACKGROUND We have reported previously that YB-1 induces translation of GluR2 mRNA in response to neural activity, and that HSP60 affects the association of YB-1 with polysomes. Here we examined the mechanism of YB-1-mediated translational activation of GluR2 mRNA through the nAChR. METHODS Expression of nAChRs in NG108-15 cells was verified. Translation of GluR2 mRNA and YB-1/HSP60 interaction were examined in nicotine-treated NG108-15 cells. Effects of inhibition of α7-nAChR and the PI3K/Akt pathway were investigated. The ratios of YB-1 to GluR2 mRNA and to HSP60 were explored in polysomal and non-polysomal fractions, respectively, and the role of HSP60 in cytoplasmic retention of YB-1 was evaluated. RESULTS Nicotine treatment transiently induced translation of GluR2 mRNA and Akt phosphorylation with a concomitant increase of YB-1/HSP60 interaction. Both α-bungarotoxin and LY294002 abolished the effects of nicotine. On a sucrose gradient, nicotine treatment shifted the distribution of YB-1 to much heavier-sedimenting polysome fractions. In these fractions, the ratio of YB-1 to its binding GluR2 mRNA was decreased, and ribosome association with the YB-1-bound GluR2 mRNA was increased. HSP60 was distributed only in the non-polysomal fractions as its binding to YB-1 increased. In HSP60-depleted cells, nicotine treatment induced nuclear localization of YB-1. CONCLUSION YB-1 is released from GluR2 mRNA during α7-nAChR-mediated neurotransmission, causing the PI3K/Akt pathway to recruit ribosomes into the translational machinery, and HSP60 is involved in cytoplasmic retention of polysome-free YB-1. GENERAL SIGNIFICANCE Activation of the PI3K/Akt pathway through the α7-nAChR and YB-1/HSP60 interaction are important for YB-1-mediated translational activation of GluR2 mRNA.

Translational level of acetylcholine receptor α mRNA in mouse skeletal muscle is regulated by YB-1 in response to neural activity

Y-box-binding protein 1 (YB-1) binds to mRNAs and affects translation. In this study, we focused on skeletal muscle, in which YB-1 expression is restricted to the early postnatal period, and found that YB-1 binds to acetylcholine receptor α-subunit (AChR α) mRNA. Although transcription of the AChR α gene is known to be regulated by myogenic transcription factors, translational control of the mRNA in response to neuromuscular transmission has not been examined. In undifferentiated C2C12 myoblasts, expression of AChR α remained at a low level. However, translation of the mRNA was increased by knockdown of YB-1. Continued overexpression of YB-1 prevented the cells from differentiating. In myotubes, which show clustering of AChRs, translation of the mRNA was induced within 3h after treatment with nicotine. The effect of nicotine was inhibited by α-bungarotoxin, and in the presence of cycloheximide the level of AChR α was reduced, even after nicotine treatment. Sucrose gradient analysis revealed that in nicotine-treated myotubes, YB-1-containing polysomes were shifted to the heavier-sedimenting fractions, and showed an apparent decrease in the amount of YB-1 bound to AChR α mRNA. These results suggest that in skeletal muscle cells, neural activity reduces the molar ratio of YB-1 relative to its binding AChR α mRNA, leading to an increase of ribosome binding to the mRNA, and thus activating translation. Furthermore, in postnatal growing mice, as has already been shown, the level of AChR α mRNA declined during the early period with maturation of neuromuscular synapses, but the translation level was found to increase transiently at postnatal day 10, when the level of YB-1 was markedly reduced. It is suggested that although the level of AChR α mRNA is reduced, the translation can be induced by alteration of the ratio of YB-1 protein to the mRNA.

C9orf10 Protein, a Novel Protein Component of Purα-containing mRNA-protein Particles (Purα-mRNPs): Characterization of Developmental and Regional Expressions in the Mouse Brain

Purα has been implicated in mRNA transport and translation in neurons. We previously reported that Purα is a component of mRNA/protein complexes (Purα-mRNPs) with several other proteins. Among them, we found the C9orf10 (Homo sapiens chromosome 9 open reading frame 10) protein, which was recently characterized as a component of RNA-containing structures. However, C9orf10 itself remains poorly understood. To characterize C9orf10 expression at the protein level, we raised an antibody against C9orf10 and compared the spatial and developmental expressions of this protein and Purα in the mouse brain. C9orf10 was expressed as early as embryo stage 12, whereas Purα was expressed from 5 days after birth. In adults, C9orf10 expression was most prominent in the hippocampus, caudate putamen, cerebral cortex, and cerebellum, unlike the uniform distribution of Purα. C9orf10-positive cells also showed immunoreactivity to Purα. C9orf10 expression was restricted to neurons, judging by the immunoreactivity to neuron-specific nuclear protein or CaM kinase II. These observations suggest an accessory role of C9orf10 for Purα in a limited brain region in addition to other possible functions that have not yet been determined.

Four nucleocytoplasmic-shuttling proteins and p53 interact specifically with the YB-NLS and are involved in anticancer reagent-induced nuclear localization of YB-1.

In cancer cells, anticancer reagents often trigger nuclear accumulation of YB-1, which participates in the progression of cancer malignancy. YB-1 has a non-canonical nuclear localization signal (YB-NLS). Here we found that four nucleocytoplasmic-shuttling RNA-binding proteins and p53 interact specifically with the YB-NLS and co-accumulate with YB-1 in the nucleus of actinomycin D-treated cells. To elucidate the roles of these YB-NLS-binding proteins, we performed a dominant-negative experiment in which a large excess of YB-NLS interacts with the YB-NLS-binding proteins, and showed inhibitory effects on actinomycin D-induced nuclear transport of endogenous YB-1 and subsequent MDR1 gene expression. Furthermore, the YB-NLS-expressing cells were also found to show increased drug sensitivity. Our results suggest that these YB-NLS-associating proteins are key factors for nuclear translocation/accumulation of YB-1 in cancer cells.

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.

YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation.