PRE-MRNA SPLICE SITE RECOGNITION IN HUMAN DISEASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们研究的总体目标是了解指导前mRNA剪接因子与3？“剪接位相互作用的三维形状，从而为开发与剪接位点识别错误相关的遗传病的治疗提供基础。U2AF是人类必不可少的剪接因子，在剪接位点选择的关键早期阶段与前mRNA 3？”剪接位的共识序列结合，并招募核心剪接机制。我们最近在同步辐射的帮助下确定了U2AF与多聚尿苷的络合物的结构[Sickmier等人。(2006)莫尔。单元格)。这种结构证实了U2AF识别尿苷的机制。然而，U2AF的体内RNA靶标含有胞嘧啶。这项拟议工作的目标是了解U2AF如何改变构象以识别胞嘧啶而不是尿嘧啶。为此，我们用三种不同的富含尿苷的寡核苷酸结晶了U2AF，这些寡核苷酸的单一位置变成了胞嘧啶。特定的核苷酸被溴化以明确地识别核酸注册表。具有其中一个序列的共晶衍射到2.9？分辨率，另外两个共晶在内部衍射到高于3.5？分辨率。需要同步辐射才能获得高分辨率的数据集，以揭示与靶RNA序列结合的这一基本剪接因子的微妙构象变化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of our research is to understand the three-dimensional shapes that guide interaction of pre-mRNA splicing factors with the 3¿¿" splice site, thereby providing a foundation to develop treatments for inherited diseases associated with errors in splice site recognition. The essential human splicing factor, U2AF binds to the pre-mRNA 3¿¿" splice site consensus sequence during the critical early stages of splice site choice, and recruits the core splicing machinery. We recently determined the structure of U2AF in complex with poly-uridine with the aid of synchrotron radiation [Sickmier et al. (2006) Mol. Cell). This structure demonstrates the mechanism of uridine recognition by U2AF. However, the in vivo RNA targets of U2AF contain cytosine. The goal of the proposed work is to understand how U2AF changes conformation to recognize a cytosine instead of uracil. To this end we have crystallized U2AF with three different uridine-rich oligonucleotides with single-positions changed to cytosine. Specific nucleotides are brominated to unambiguously identify the nucleic acid register. Cocrystals with one of the sequences diffract to 2.9¿ resolution, and two other cocrystals diffract to better than 3.5¿ resolution in-house. Synchrotron radiation is required to obtain the high resolution data sets to reveal subtle conformational changes of this essential splicing factor bound to target RNA sequences.