PRE-MRNA SPLICE SITE RECOGNITION IN HUMAN DISEASE

人类疾病中的前 mRNA 剪接位点识别

• 批准号: 7955565
• 负责人: CLARA KIELKOPF
• 金额: $ 0.46万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955565
• 关键词: Binding; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Consensus Sequence; Cytosine; Data Set; Disease; Foundations; Funding; Goals; Grant; Housing; Human; Inherited; Institution; Molecular Conformation; Nucleic Acids; Nucleotides; Oligonucleotides; Positioning Attribute; RNA; RNA Sequences; RNA Splicing; Recruitment Activity; Research; Research Personnel; Resolution; Resources; Shapes; Site; Source; Staging; Structure; United States National Institutes of Health; Uracil; Uridine; Work; human disease; in vivo; mRNA Precursor; synchrotron radiation

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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 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剪接机械。我们最近在同步辐射的帮助下确定了与多尿尿素复合物中U2AF的结构[Sickmier等。 （2006）摩尔。细胞）。该结构证明了U2AF识别尿苷的机制。但是，U2AF的体内RNA靶标含有胞嘧啶。拟议工作的目的是了解U2AF如何改变构象以识别胞嘧啶而不是尿嘧啶。为此，我们用三个不同的尿苷寡核苷酸结晶了U2AF，单位固定核苷酸转换为胞嘧啶。特定的核动肽被溴化以明确识别核酸登记量。与一个序列之一衍射为2.9的共晶，另外两个共晶衍射效果比内部分辨率更好。需要同步加速器辐射以获得高分辨率数据集，以揭示与目标RNA序列结合的基本剪接因子的微妙构象变化。