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: 8363522
• 负责人: CLARA KIELKOPF
• 金额: $ 0.57万
• 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/8363522
• 关键词: 3' Splice Site; Affinity; Binding; Cells; Complement; Complex; Consensus Sequence; Cystic Fibrosis; Cytidine; Data; Dimensions; Disease; Funding; Goals; Grant; Human; Hydrogen Bonding; Inherited; Malignant Neoplasms; Measures; Mediating; Metabolic Diseases; Methods; Modeling; Molecular; Muscular Dystrophies; National Center for Research Resources; Nucleotides; Principal Investigator; RNA; RNA Recognition Motif; RNA Splicing; Recruitment Activity; Relative (related person); Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Sampling; Series; Shapes; Side; Site; Solutions; Source; Staging; Structure; United States National Institutes of Health; Uridine; Water; Work; base; beamline; cost; flexibility; 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. 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 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. These structures would contribute to understanding the basis for many inherited diseases that are associated with errors in splice site recognition, including cancers, metabolic disorders, muscular dystrophies, and cystic fibrosis among others. The essential splicing factor, U2AF binds to the pre-mRNA 3' splice site consensus sequence during the critical early stages of splice site choice, and recruits further components of the splicing machinery. The pre-mRNA sequence recognized by U2AF is a polypyrimidine tract composed primarily of uridines and cytidines. A few years ago, we determined the structure of U2AF bound to an optimal polyuridine splice site with the aid of synchrotron radiation [Sickmier et al. (2006) Mol. Cell]. This structure demonstrates that U2AF recognizes uridines through hydrogen bonds with the edges of the bases. However, the in vivo RNA targets of human U2AF are frequently interrupted by cytidines. To investigate how the U2AF structure adapts to accommodate cytidines, we determined a series of U2AF bound to cytidine-containing RNAs using the microfocus F1 beamline at CHESS. From these structures, we concluded that U2AF (1) rearranges side-chain and water-mediated hydrogen bonds and (2) adjusts its binding register to adapt to cytidine-containing sites. We complemented these crystallographic results by measuring the affinities of U2AF for the cytidine-containing site, and found that U2AF can tolerate up to four consecutive cytidines in the polypyrimidine tract with little difference among affinities, but beyond this limit can no longer adapt. The polypyrimidine tract-binding domain of U2AF contains two modular RNA recognition motifs (RRMs) separated by a poorly conserved interdomain linker. By using the ensemble optimization method (EOM) to analyze small angle X-ray scattering data collected at the SIBYLS beamline at ALS, we found that the relative arrangement of the U2AF RRMs appears to be flexible. Based on these data, we hypothesize that in solution, the flexible RRM arrangement contributes to the ability of U2AF to adapt to variable splice sites. Here, we propose to compare the low resolution shapes of U2AF complexes with (1) a natural polypyrimidine tract sequence (CCCUUUUUUUUCC), (2) the polyuridine site (UUUUUUUUUUUUU), and (3) a sequence that represents the limit of cytidine-substitutions that U2AF binds without detectable decrease in affinity (CCCUUUCUCCUCC) using small angle X-ray scattering at CHESS beamline G1. Sequence-dependent changes in the molecular dimensions of the complexes would support the hypothesis that adjustable inter-RRM register contributes to U2AF ability to adapt to cytidine-containing sequences. All samples are monodisperse. SAXS data was previously collected for an analogous complex of U2AF with a 12-nucleotide polyuridine RNA at the SIBYLS beamline at ALS. This data produced consistent Guinier plots over a concentration range (2-8 mg/mL) and resulted in Chi values <2 when modeled using Dammin. This work is funded by the National Institutes of Health, R01-GM 070503.

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 子项目的主要研究者可能是由其他来源提供的， 包括其他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。 我们研究的总体目标是了解引导前体mRNA剪接因子与3'剪接位点相互作用的三维形状。这些结构将有助于理解许多与剪接位点识别错误相关的遗传性疾病的基础，包括癌症、代谢紊乱、肌营养不良和囊性纤维化等。 在剪接位点选择的关键早期阶段，必需剪接因子U2 AF与前体mRNA 3'剪接位点共有序列结合，并募集剪接机制的其他组分。由U2 AF识别的前mRNA序列是主要由尿苷和胞苷组成的多聚嘧啶段。几年前，我们借助于同步辐射确定了与最佳聚尿苷剪接位点结合的U2 AF的结构[Sicklett等人（2006）Mol. Cell]。 该结构表明U2 AF通过与碱基边缘的氢键识别尿苷。然而，人U2 AF的体内RNA靶标经常被胞苷中断。为了研究U2 AF结构如何适应容纳胞苷，我们使用CHESS的微焦点F1光束线确定了一系列与含胞苷RNA结合的U2 AF。从这些结构中，我们得出结论，U2 AF（1）重排侧链和水介导的氢键和（2）调整其结合寄存器，以适应含胞苷的网站。我们通过测量U2 AF对含胞苷位点的亲和力来补充这些晶体学结果，并发现U2 AF可以耐受多聚嘧啶道中最多四个连续的胞苷，亲和力之间几乎没有差异，但超过这个限度就不能再适应了。 U2 AF的多聚嘧啶片段结合结构域包含两个由保守性差的结构域间连接子分开的模块化RNA识别基序（RRM）。通过使用系综优化方法（EOM）来分析在ALS的SIBYLS光束线处收集的小角X射线散射数据，我们发现U2 AF RRM的相对布置似乎是灵活的。基于这些数据，我们假设在溶液中，灵活的RRM安排有助于U2 AF适应可变剪接位点的能力。在这里，我们建议使用CHESS光束线G1处的小角X射线散射，将U2 AF复合物的低分辨率形状与（1）天然聚嘧啶序列（CCCUUUUUUUUCC），（2）聚尿苷位点（UUUUUUUUUUUU）和（3）代表U2 AF结合而亲和力无可检测降低的胞苷取代极限（CCCUUUCUCCUCC）的序列进行比较。复合物分子尺寸的序列依赖性变化将支持可调节的RRM间寄存器有助于U2 AF适应含胞苷序列的能力的假设。 所有样品都是单分散的。SAXS数据先前收集的类似复合物的U2 AF与12个核苷酸的聚尿苷RNA在SIBYLS光束线在ALS。该数据在浓度范围（2-8 mg/mL）内产生一致的Guinier图，并在使用Dammin建模时产生<2的Chi值。 这项工作由美国国立卫生研究院（R 01-GM 070503）资助。