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

临床上重要的抗肿瘤剂5-氟尿嘧啶（5-FU）是 旨在抑制胸苷酸合成酶（TS）。TS的有效抑制作用 是5-FU疗效的一个重要方面，但长期以来， 越来越多的证据表明，5-FU干扰核糖核酸（RNA） 介导的过程，特别是前体信使RNA的剪接 （前mRNA）。RNA介导的5-FU作用可能构成了一种新的靶点。 它的大部分力量。插入序列的切除， 外显子的剪接发生在剪接体中，剪接体是一种小的 核RNA（snRNA）和相关蛋白。双工由单个 两种或更多种不同类型的snRNA的链区出现在 剪接体的形成。snRNA之间的这种关联是基于 在互补区域的碱基配对上， 前体mRNA的剪接。 这些双链体含有鸟苷-尿苷（GU） 和尿苷-尿苷（UU）摆动碱基对，以及鸟苷- 胞苷（GC）和腺苷-尿苷（Au）碱基对。的氟取代 在U的C5处的变化可能会影响摆动和正常碱基的相对稳定性 配对并改变含有5-FU的snRNA复合物的结合稳定性。 5-FU掺入RNA的结构结果并不好 目前了解的RNA和建议的分析含有5-FU的RNA， 核磁共振（NMR）光谱将有助于我们 RNA指导的这种药物的效力和毒性机制的知识， 药该提案旨在将5-氟尿苷掺入RNA低聚物中 使用溶液和固相化学物质。U4-U6的详细模型 snRNA复合物基于NMR光谱数据开发。两个茎 研究了由人U4和U6 snRNA相互作用形成的区域， 详细通过使用1H NMR光谱。5-氟尿苷的影响 对每种尿苷的取代进行化学评价。 双链 用~ 1H NMR对具有反常熔化曲线的三种金属化合物进行了详细的研究 谱U4-U6 snRNA复合物的一部分是通过使用一种 体外T7 RNA聚合酶转录系统。同位素富集 将三磷酸核苷（NTP）掺入U4-U6 snRNA中 络合物，然后通过1H-13 C和1H-15 N NMR光谱分析。的 5-FU掺入对U4-U6 snRNA复合物的总体影响是 用光谱法评价。 了解结构基础， RNA介导的5-FU的效力和毒性将导致 合理干预前mRNA的未来抗癌策略 剪接或其它RNA介导的细胞过程。

The clinically important anti-tumor agent 5-fluorouracil (5-FU) was designed to inhibit thymidylate synthase (TS). The potent inhibition of TS is an important aspect of the efficacy of 5-FU, but there is longstanding and growing evidence that 5-FU interferes with ribonucleic acid (RNA) mediated processes, particularly the splicing of precursor messenger RNA (pre-mRNA). It is possible that RNA directed actions of 5-FU constitute a major portion of its potency. The excision of intervening sequences and splicing of exons takes place in the spliceosome - a complex of small nuclear RNAs (snRNA) and associated proteins. Duplexes composed of single stranded regions of two or more different types of snRNA occur upon formation of the spliceosome. This association between snRNAs is based upon base pairing of complementary regions and is required for proper splicing of the pre-mRNA. These duplexes contain guanosme-uridine (GU) and uridine-uridine (UU) wobble base pairs in addition to guanosine- cytidine (GC) and adenosine-uridine (AU) base pairs. Fluorine substitution at C5 of U may impact the relative stability of wobble and normal base pairs and alter the binding stability of snRNA complexes containing 5-FU. The structural consequences of 5-FU incorporation into RNA are not well understood at present and the proposed analysis of RNA containing 5-FU by nuclear magnetic resonance (NMR) spectroscopy will contribute to our knowledge of the RNA directed mechanisms of potency and toxicity of this drug. This proposal aims to incorporate 5-fluorouridine into RNA oligomers using solution and solid-phase chemistries. A detailed model of the U4-U6 snRNA complex is developed based upon NMR spectroscopic data. The two stem regions formed by the interaction of human U4 and U6 snRNAs are studied in detail by using 1H NMR spectroscopy. The impact of 5-fluorouridine substitution for each uridine is assessed thermodynamically. Duplexes with anomalous melting profiles are studied in detail by using 1H NMR spectroscopy. A portion of the U4-U6 snRNA complex is prepared by using an in vitro T7 RNA polymerase transcription system. Isotopically enriched nucleoside triphosphate (NTP) is incorporated into the U4-U6 snRNA complex, which is then analyzed by 1H-13C and 1H-15N NMR spectroscopy. The overall impact of 5-FU incorporation on the U4-U6 snRNA complex is evaluated spectroscopically. Understanding the structural basis for the RNA mediated potency and toxicity of 5-FU will ald in the development of future anticancer strategies that rationally intervene in pre-mRNA splicing, or other RNA mediated cellular processes.