STUDY OF HCV RNA-PROTEIN INTERACTIONS

HCV RNA-蛋白质相互作用的研究

• 批准号: 8361510
• 负责人: Charles M Rice
• 金额: $ 0.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361510
• 关键词: Beryllium; Cells; Elements; Funding; Gel; Grant; Hepatitis C virus; Hepatocyte; Human; In Vitro; National Center for Research Resources; Oligonucleotides; Principal Investigator; Proteins; RNA; RNA Interference; RNA replication; RNA-Binding Proteins; RNA-Protein Interaction; Research; Research Infrastructure; Resources; Role; Source; Streptavidin; Time; United States National Institutes of Health; cost; follow-up; knock-down; macromolecule; magnetic beads; mutant; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The aim of the project is to isolate and identify host proteins that interact with the HCV RNA structural element. The in vitro transcribed RNA element was first annealed to the biotinylated oligonucleotide, then was immobilized to the streptavidin magnetic beads. The soluble fraction (cytoplasmic) from Human Hepatocyte cell lysates was passed over with the RNA conjugated magnetic beads. After washing several times, the final pull-out was analyzed on SDS-PAGE gel, followed by MS and MS/MS analyses. We have performed comparisons between isolations using non-specific oligo, 320nt, or CRE and Specific 3'NTR, Cre-3"NTR, and Cre-3'NTR using Huh7.5 or Flneo cells. Competition with wild or mutant RNA helped to distinguish interacting proteins specific to the RNA. From the MS/MS analyses, we identified several RNA binding proteins. Follow-up with RNAi knock down analysis indicates that two identified proteins, LRP130 and DDX3, seems to be related with the HCV RNA replication. Further experiments will be followed to understand their roles in the HCV replication.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 该项目的目的是隔离和鉴定与HCV RNA结构元件相互作用的宿主蛋白。首先将体外转录的RNA元件退火到生物素化的寡核苷酸，然后固定在链霉亲蛋白 磁珠。人肝细胞裂解物的可溶性馏分（细胞质）用RNA偶联的磁珠传递。洗涤几次后，对SDS-PAGE凝胶进行了分析，然后分析了MS和MS/MS分析。我们已经使用非特异性寡聚，320NT或CRE和特定的3'NTR，CRE-3“ NTR和使用HUH7.5或Flneo细胞的CRE-3'NTR进行了比较。与野生或突变RNA的竞争有助于区分与rna的相互作用的蛋白质，从而确定了分析的蛋白质，我们确定了几个rna rna的竞争。鉴定出的蛋白质LRP130和DDX3似乎与HCV RNA复制有关，以了解其在HCV复制中的作用。