IMPACT OF 5FU ON THE STRUCTURE OF THE U4/U6 COMPLEX

描述：我们的知识中最大的空白之一是如何 广泛使用的抗癌药物5-氟尿嘧啶(5-FU)干扰RNA介导的作用 过程是蛋白质-RNA相互作用在多大程度上受到干扰 尿苷(S)(URD)在蛋白质识别位点(S)的核糖核酸是 替换为FURD。在此应用程序中，将检验该假设 U4SnRNA富含Urd的Sm结合位点上的FURD替换破坏 SnRNP核心蛋白(Sm蛋白：B、D_1、D_2、 D3、E、F和G)，一组以其亲缘关系命名的碱性蛋白质 十二烷基硫酸钠期间的迁移率-PAGE。每个剪接体SnRNA(U6除外 SnRNA)包含一个Sm结合位点(S.cerevisae中的共同序列= AAUUUUGG)。U4单链RNA不能与Sm复合体结合会降低 U4核糖核糖核酸的转运效率和抑制形成 剪接体。最近测量突变的生物效应的研究 在U4中，SnRNA表明几乎没有耐受性 Sm结合位点，建议采用刚性识别码 Sm蛋白复合体和Sm结合部位。将确定是否 U4单链RNA Sm结合位点处及附近的FURD替换破坏 U4单链RNA这一区域的结构和稳定性以及它是否影响 U4单链RNA与Sm蛋白的络合作用。这将通过以下方式完成：1) U4单链RNA 3‘端三维结构的测定 多维核磁共振波谱联合应用于酿酒酵母的研究 分子模拟；2)评估FURD取代对 U4单链RNA 3‘端结构、稳定性和动力学的研究 核磁共振波谱、核酸酶探测和紫外光增色研究；3) 证明U4的Sm结合位点的单核苷酸突变 SnRNA通过以下方式降低RNA对Sm蛋白复合体的亲和力 ~(32)P标记的酵母培养的U4单链RNA的免疫印迹 Sm蛋白的特异性抗体Y12的胞浆提取液。 4)证明了Sm结合中的FURD取代 U4单链RNA的位点也破坏RNA-蛋白质复合物的形成 免疫印迹程序。这些研究将提供新的信息 关于U4单链RNA 3‘区的天然结构， 天然核苷酸替代对Sm络合物形成的影响 FURD取代对U4单链RNA及其络合物结构和稳定性的影响 含Sm蛋白。

DESCRIPTION: One of the largest gaps in our knowledge concerning how the widely used anticancer drug 5-fluorouracil (5-FU) disrupts the RNA mediated processes is to what extent protein-RNA interactions are perturbed when uridine (s) (Urd) in the protein recognition site(s) of the RNA are substituted with FUrd. In this application the hypothesis will be tested that FUrd substitution in the Urd-rich Sm binding site of U4 snRNA disrupts recognition of U4 snRNA by the snRNP core proteins (Sm proteins: B, D1, D2, D3, E, F, and G), a collection of basic proteins named for their relative mobilities during SDS-PAGE. Each of the spliceosomal snRNAs (except U6 snRNA) contains an Sm binding site (consensus sequence in S. cerevisae = AAUUUUUGG). Failure of U4 snRNA to bind to the Sm complex would reduce the efficiency of U4 snRNA nuclear transport and inhibit formation of the spliceosome. Recent studies measuring the biological effects of mutations in U4 snRNA indicate virtually no tolerance for nucleotide substitution in the Sm binding site, suggesting adoption of a rigid recognition code between the Sm protein complex and the Sm binding site. It will be determined if FUrd substitution at and near the Sm binding site of U4 snRNA disrupts the structure and stability of this region of U4 snRNA or if it affects complexation of U4 snRNA with Sm proteins. This will be done by: 1) determining the three dimensional structure for the 3' region of U4 snRNA from S. cerevisae using multidimensional NMR spectroscopy in conjunction with molecular modeling; 2) assessing the effects of FUrd substitution on the structure, stability, and dynamics of the 3' region of U4 snRNA using NMR spectroscopy, nuclease probing, and UV hyperchromicity studies; 3) demonstrating that single nucleotide mutations in the Sm binding site for U4 snRNA decrease the affinity of the RNA for the Sm protein complex by immunoblotting 32P- labeled U4 snRNA that has been incubated with yeast cytosolic extract with Y12, an antibody specific for Sm protein from S. cerevisae; and 4) demonstrating that FUrd substitution in the Sm binding site of U4 snRNA disrupts RNA-protein complex formation also using immunoblotting procedures. These studies will provide novel information concerning the native structure of the 3' region of U4 snRNA, the effects of native nucleotide substitution on Sm complex formation and the effects of FUrd substitution on the structure and stability of U4 snRNA and its complex with Sm proteins.