PRE-MRNA SPLICE SITE RECOGNITION IN HUMAN DISEASE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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遗传性疾病的基础，这些疾病与剪接位点识别错误有关，包括癌症、代谢障碍、肌肉营养不良和囊性纤维化等。 重要的剪接因子U2AF在剪接位点选择的关键早期阶段与前mRNA 3‘剪接位点共识序列结合，并招募剪接机械的进一步组件。U2AF识别的Pre-mRNA序列是一个主要由尿苷和胞苷组成的多嘧啶核苷链。几年前，我们在同步辐射的帮助下，确定了U2AF与最佳多聚尿苷剪接位点结合的结构。(2006)莫尔。细胞]。这种结构表明U2AF通过与碱基边缘的氢键识别尿苷。然而，人U2AF的体内RNA靶点经常被胞苷打断。为了研究U2AF结构如何适应胞苷，我们在CHESS用微焦点F1光束线确定了一系列与胞苷RNA结合的U2AF。根据这些结构，我们得出结论，U2AF(1)重排侧链和水介导的氢键，(2)调整其结合寄存器以适应含胞苷的位置。我们通过测量U2AF与胞苷结合位点的亲和力来补充这些结晶学结果，发现U2AF可以在多嘧啶途径中耐受多达四个连续的胞苷，亲和力之间几乎没有差异，但超过这个极限就不能再适应了。 U2AF的多嘧啶结合域包含两个模块化的RNA识别基序(RRMS)，由一个保守的域间连接子隔开。通过使用系综优化方法(EOM)分析在ALS的SIBYLS光束线上收集的小角X射线散射数据，我们发现U2AF RRMS的相对排列是灵活的。基于这些数据，我们假设在解决方案中，灵活的RRM排列有助于U2AF适应可变剪接位点的能力。在这里，我们建议比较U2AF复合体的低分辨率形状与(1)天然多嘧啶通道序列(CCCUUUUUUCC)，(2)多尿苷位点(UUUUUUUUUUUUUU)，以及(3)表示U2AF结合的胞苷取代极限的序列(CCCUUCUCCUCC)，该序列表示U2AF结合的胞苷取代的极限而没有检测到亲和力的降低(CCCUUCUCCUCC)。复合体分子尺寸的序列依赖性变化将支持这样的假设，即可调节的RRM间寄存器有助于U2AF适应含胞苷的序列。 所有样品都是单分散的。SAXS数据之前是在ALS的SIBYLS光束线收集的U2AF与12核苷酸多尿苷RNA的类似复合体。这些数据在浓度范围内(2-8 mg/mL)产生了一致的Guinier曲线，并在使用Dammin建模时得出了CHI值&lt；2。 这项工作由美国国立卫生研究院资助，R01-GM 070503。

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.