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: 2101357
• 负责人: William H Gmeiner
• 金额: $ 10.45万
• 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/2101357
• 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 旨在抑制胸苷酸合酶（TS）。强效抑制TS 是5-FU疗效的一个重要方面，但长期以来存在 越来越多的证据表明 5-FU 会干扰核糖核酸 (RNA) 介导的过程，特别是前体信使RNA的剪接 （前mRNA）。 5-FU 的 RNA 定向作用可能构成 其效力的主要部分。干扰序列的切除和 外显子的剪接发生在剪接体中——剪接体是一个小分子的复合体 核 RNA (snRNA) 和相关蛋白。复式公寓由单层公寓组成 两种或多种不同类型 snRNA 的绞合区发生在 剪接体的形成。 snRNA 之间的这种关联是基于 基于互补区域的碱基配对，并且是正确的 前体 mRNA 的剪接。 这些双链体含有鸟苷-尿苷 (GU) 除了鸟苷之外，还有尿苷-尿苷 (UU) 摆动碱基对 胞苷 (GC) 和腺苷-尿苷 (AU) 碱基对。氟取代 U的C5处可能会影响摆动和正常底座的相对稳定性 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 的结构后果并不理想 目前的理解和建议的含有 5-FU 的 RNA 分析 核磁共振（NMR）光谱将有助于我们 RNA 指导的效力和毒性机制的知识 药品。该提案旨在将 5-氟尿苷掺入 RNA 寡聚物中 使用溶液和固相化学。 U4-U6的详细模型 snRNA 复合物是根据 NMR 光谱数据开发的。两个茎 研究了人类 U4 和 U6 snRNA 相互作用形成的区域 通过使用 1H NMR 光谱进行详细分析。 5-氟尿苷的影响 通过热力学评估每个尿苷的取代。 复式公寓 使用 1H NMR 详细研究了具有异常熔化曲线的物质 光谱学。 U4-U6 snRNA 复合物的一部分是通过使用 体外 T7 RNA 聚合酶转录系统。同位素富集 三磷酸核苷 (NTP) 掺入 U4-U6 snRNA 中 络合物，然后通过 1H-13C 和 1H-15N NMR 光谱进行分析。这 5-FU 掺入对 U4-U6 snRNA 复合物的总体影响是 通过光谱评估。 了解结构基础 RNA 介导的 5-FU 的效力和毒性将有助于开发 合理干预前体mRNA的未来抗癌策略 剪接或其他 RNA 介导的细胞过程。