LOCATING THE REGIONS OF AN ADML 3' INTRON RNA ANALOGUE BOUND TO PUF60 RRMS

定位与 PUF60 RRMS 结合的 ADML 3 内含子 RNA 类似物的区域

• 批准号: 8363546
• 负责人: DEMETRIOS BRADDOCK
• 金额: $ 0.45万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363546
• 关键词: Affinity; Alternative Splicing; Binding; Bromine; Complex; Dissociation; Exons; Funding; Grant; Homo; Human Genome; Introns; Mediating; Modeling; Modification; National Center for Research Resources; Nucleic Acids; Pattern; Principal Investigator; RNA; RNA Processing; RNA-Binding Proteins; Research; Research Infrastructure; Resources; Source; Specific qualifier value; Spliceosome Assembly Pathway; Staging; Structure; United States National Institutes of Health; analog; base; beamline; cost; dimer; electron density; mRNA Precursor; monomer

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. Alternative splicing is a major mechanism for RNA processing which significantly contributes to the expanded complexity of the expression of human genome. In metazoan, the early-stage recognition of the 3' pre-mRNA intron sequence by RNA binding proteins (RNABPs) is considered to be essential for specifying intron/exon- definitions and priming the pre-mRNA for the later stages of spliceosome assembly. PUF60 is such an RNABP which may promote alternative splicing patterns. We determined the dissociation constants of PUF60 toward the AdML 3' intron sequence to be at the uM-range, with the binding affinity predominantly provided by its two central RRMs. We have also obtained a 1.91 A crystal structure of the two RRMs of PUF60 in complex with AdML3' intron sequence, which reveals a homo-dimeric organization, where each participating PUF60 monomer captures a nucleic acid tract, and the PUF60 dimer places two nucleic acid tracts in parallel with opposing directionalities. However, where PUF60 RRMs bind on the RNA analogue has not been located because two of the bound bases have poor electron densities. Therefore, we have grown new crystals of PUF60 RRMs in complex with Bromine-modified RNA analogues, with the aim to identify the bound bases. CHESS beamlines hold the potential to help us visualize the Bromine modification on our RNA analogues, and therefore allow us to locate the bound regions and propose a more precise model for the PUF60 RRMs mediated RNA interactions.

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。 替代剪接是RNA加工的主要机制，它显着有助于人类基因组表达的复杂性扩大。在后生动物中，RNA结合蛋白（RNABPS）对3'前MRNA内含子序列的早期识别被认为对于指定内含子/外显子定义并为剪接组组件的后期启动MRNA至关重要。 PUF60是这样的RNABP，可以促进替代剪接模式。 我们确定了PUF60对ADML 3'内含子序列的解离常数为在UM范围内，其结合亲和力主要由其两个中央RRM提供。我们还获得了与Adml3'内含子序列复合物中PUF60的两个RRM的1.91 A晶体结构，该序列揭示了一个同性含量的组织，每个组织都参与PUF60单体捕获核酸区域，PUF60二聚体将两个核酸放置与相反方向并行的区域。但是，由于puf60 rrm的结合在RNA类似物上的情况下，由于两个结合碱基的电子密度较差，因此尚未找到RNA模拟。因此，我们与溴化的RNA类似物中的PUF60 RRM的新晶体成长为复合物，目的是识别结合碱基。国际象棋束线条具有帮助我们可视化RNA类似物的溴修饰的潜力，因此使我们能够找到结合区域，并为PUF60 RRMS介导的RNA相互作用提出了更精确的模型。