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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 选择性剪接是RNA加工的主要机制之一，它极大地增加了人类基因组表达的复杂性。在后生动物中，RNA结合蛋白（RNABP）对3'前mRNA内含子序列的早期识别被认为是指定内含子/外显子定义和启动剪接体组装后期阶段的前mRNA所必需的。PUF 60是这样一种RNABP，它可以促进选择性剪接模式。 我们测定了PUF 60对AdML 3'内含子序列的解离常数在μ M范围内，结合亲和力主要由其两个中心RRM提供。我们还获得了与AdML 3 '内含子序列复合的PUF 60的两个RRM的1.91 A晶体结构，其揭示了同源二聚体组织，其中每个参与的PUF 60单体捕获核酸束，并且PUF 60二聚体将两个核酸束以相反的方向平行放置。然而，PUF 60 RRM在RNA类似物上结合的位置尚未定位，因为结合的碱基中的两个具有差的电子密度。因此，我们已经生长了与溴修饰的RNA类似物复合的PUF 60 RRM的新晶体，目的是鉴定结合的碱基。CHESS光束线有可能帮助我们可视化RNA类似物上的溴修饰，因此使我们能够定位结合区域并为PUF 60 RRM介导的RNA相互作用提出更精确的模型。

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.