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题目:: RNA binding of mouse retinal ganglion cells (RGCs) in multiple sclerosis and in glaucoma eyes compared to normal controls to identify RNA species bound by deiminated and non-deiminated MA-Brent-1 (bhatt-affy-mouse-581641)
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状态:: 发布时间July 1, 2009 , 更新时间 June 10, 2011 , 提交时间 June 20, 2008,
物种:: Mus musculus
摘要:: We have identified loss of deiminated MA-Brent-1 (an RNA and export binding protein) in the retinal ganglion cells (RGCs) in multiple sclerosis and in glaucoma eyes compared to normal controls. Deimination refers to posttranslational modification of protein bound arginine (not free arginine) in citrulline. Our preliminary studies suggest binding of different repertoire of RNA by non-deiminated and deiminated MA-Brent-1. In vitro, in neurites of cultured RGCs and hippocampal neurons, the select mRNA translation is enhanced by addition of deiminated but not non-deiminated MA-Brent-1. These observations suggest that lack of deiminated MA-Brent-1 has consequences for protein synthesis, remodeling and plasticity of RGCs/neurons. Identification of RNA species bound by deiminated and non-deiminated MA-Brent-1 will enable us there further verification and determining the role that deimination plays in biological function of MA-Brent-1 in multiple sclerosis and glaucoma. To summarize identification of RNA species bound by deiminated and non deiminated MA-Brent-1 will enable us to gain further insight into role of deimination in the overall disease process. To identify the RNA species bound by deiminated and non-deiminated MA-Brent-1; The deiminanted MA-Brent-1 binds to a different repertoire of RNA and non-deiminated MA-Brent-1. Corollary to this hypothesis is that the deiminated MA-Brent-1 enhances translation for bound species of RNA. Loss of deimination results in less dendritic protein synthesis and has consequences for remodeling and plasticity in multiple sclerosis and glaucoma RGCs where lack of deimination (compared to control eyes) has been observed. About 10 microgram of purified recombinant and his-tagged non-deiminated MA-Brent-1 or deiminated MA-Brent-1 will be incubated with about 400 microgram of total RNA. The RNA bound his-tagged protein will be incubated with about 100 microliters of Ni-NTA beads and loaded onto a mini column. The column will be washed with 50 volumes of binding buffer. The bound his-tagged protein will be released using 100 mM immidazole in the binding buffer. The MA-Brent-1 bound RNA will be recovered by boiling the protein in 0.25% SDS followed by purification of RNA using columns or using a 4% PAGE-0.8% agarose composite gel followed by gel purification. The composite gel RNA purification is routine practice in the laboratory. RNA binding experiment for each protein variant (deiminated or non-deiminated) MA-Brent-1 will be repeated twice. Identified RNA species common in both binding experiments for a given species will be candidates for further pursuit. The recombinant purified MA-Brent-1 will be subjected to deimination by incubating with recombinant peptidylarginine deiminase type II (PAD2). PAD2 is a C-terminus GST tag fusion protein and will be purified using GST column. Deiminated and non-deiminated MA-Brent-1, C-terminal his-tag fusion proteins will be purified using Ni-NTA beads/column and immidazole containing buffer as eluents. Experiment Overall Design: in vitro RNA binding with deiminated and non-deiminated MA-Brent-1
实验种类:: other
样本量:: 5
实验设计:
: 无设计数据
数据号:: E-GEOD-11843, GSE11843
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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