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

5-FU 对 U4-U6 复合物结构的影响

• 批准号: 3460804
• 负责人: William H Gmeiner
• 金额: $ 10.56万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-16 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3460804
• 关键词: RNA; RNA splicing; antineoplastics; chemical substitution; conformation; fluorouracil; neoplasm /cancer pharmacology; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; protein structure; thermodynamics; ultraviolet spectrometry; uridine

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.

临床上重要的抗肿瘤药物5-氟尿嘧啶(5-Fu)是 设计用来抑制胸苷合成酶(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 (前信使核糖核酸)。5-FU的RNA导向作用可能构成一种 其效力的主要部分。插入序列和插入序列的删除 外显子的剪接发生在剪接体--一个由小分子组成的复合体 核RNA(SnRNA)及相关蛋白。由单项组成的双面 两个或更多不同类型的SnRNA的链状区域发生在 剪接体的形成。SnRNA之间的这种关联基于 在互补区域的碱基配对上，这是正确的 前信使核糖核酸的剪接。这些双链含有鸟苷-尿苷(GU)。 和尿苷-尿苷(UU)摆动碱基对，除了鸟苷- 胞苷(GC)和腺苷-尿苷(AU)碱基对。氟取代 U的C5值可能会影响摆动和正常碱基的相对稳定性 并改变含5-FU的小分子RNA复合体的结合稳定性。 5-FU掺入RNA的结构后果并不好 目前了解的和建议的含有5-FU的RNA的分析方法 核磁共振波谱将有助于我们的 了解RNA引导的这种药物的效力和毒性机制 毒品。这项提议旨在将5-氟尿苷掺入RNA低聚物中 使用溶液和固相化学。U4-U6的详细型号 SnRNA复合体是基于核磁共振波谱数据开发的。两个茎 对人类U4和U6单链RNA相互作用形成的区域进行了研究 用~1H核磁共振波谱进行了详细的分析。5-氟尿嘧啶的影响 对每种尿苷的替代进行热力学评估。双面打印 用~1H核磁共振对异常熔化剖面进行了详细的研究 光谱学。U4-U6 SnRNA复合体的一部分通过使用 体外T7RNA聚合酶转录系统。同位素富集型 核苷三磷酸(NTP)被掺入U4-U6单链RNA中 然后用1H-13C和1H-15N核磁共振波谱对其进行分析。这个 5-FU掺入对U4-U6单链RNA复合体的整体影响 通过光谱分析进行评估。了解数据的结构基础 RNA介导的5-FU在肺癌发生发展中的效力和毒性 合理干预前信使核糖核酸的未来抗癌策略 剪接，或其他RNA介导的细胞过程。