Analysis of multifunctional property of Y-box protein in the nervous system.

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In order to investigate functions of a Y-box protein rBYB1 in cytoplasm and nucleus of the neuronal cells, we used rat brain extract, primary hippocampal neurons and nucleus of neuroblastoma NG108-15 cells. 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. Distribution of the rBYB1 in a sucrose density gradient revealed that the protein interacts with polysomes via RNA. It has been reported that Y-box proteins bind to mRNAs and modulate translation. We isolated mRNAs interacting with rBYB1 in the developing brain by immunoprecipitation using anti-rBYB1 antibody and identified some mRNAs encoding proteins that are necessary for neurite formation or neuronal transmission, such as Actin beta, Tubulin alpha 1, Glutamate receptor, and Glutamine transporter. We further found a significant change in polysome association of rBYB1 in the brain of kainic acid treated mice and decrease of rB … More YB1 levels in KCl treated primary hippocampal neurons. These observations indicate that the Y-box protein regulates the translation of these mRNAs in neuronal activity dependent manner. In the future work, we will study the role(s) of rBYB1 in the neuronal activity dependent translational control. Furthermore, we examined the expression levels and polysome-bindings of the Y-box protein in various tissues, and observed different expression patterns in developing tissues. Identification of mRNAs will be the subject of further investigation.In cultured cell lines and/or tumor cells, a Y-box protein YB1 has been reported to modulate gene expression, including that of multidrug resistance gene (MDR1). YB1 recognizes an inverted CCAAT motif (Y-box sequence) on the promoter region of MDR1 gene. Because Y-box proteins can bind to both DNA and RNA, we investigated effects of nuclear RNA on DNA-binding of rBYB1 to the MDR1 promoter and found that nuclear RNA inhibits DNA-binding of rBYB1. Interestingly, nuclear RNA also inhibits DNA-binding of NF-Y, another protein known to be a transcription factor, which recognizes a Y-box sequence and participates in MDR1 expression. We used in vitro transcription system and found that nuclear RNA repressed the transcription from MDR1 promoter. These results suggest that nuclear RNA may take part in the regulation of MDR1 gene expression in neuroblastoma through affecting DNA-binding of the Y-box protein and NF-Y. RNA molecules should be isolated from brain or analised by SELEX. Less