Dynamic Regulation of y-Globin mRNA Stability

γ-珠蛋白 mRNA 稳定性的动态调节

• 批准号: 6527871
• 负责人: J ERIC RUSSELL
• 金额: $ 27.74万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527871
• 关键词: RNase protection assay; aminobutyrate; chemical stability; developmental genetics; gene expression; gene induction /repression; genetic regulation; genetic regulatory element; genetically modified animals; globin; hydroxyurea; laboratory mouse; messenger RNA; nucleic acid structure; pharmacokinetics; posttranscriptional RNA processing; protein structure function; tissue /cell culture

DESCRIPTION: (Investigator's abstract) A comprehensive understanding of the molecular bases for the developmental stage-restricted expression of human gamma-globin is crucial to the generation of methods designed to reactivate its expression in individuals with congenital beta-chain defects. Although the fetal-to-adult globin switch is effected largely through mechanisms acting at the level of gene transcription, recent studies suggest that post-transcriptional events may also participate in this process. The current proposal is designed to investigate the possibility that physiological and pharmacological induction of gamma-globin expression may be linked to an increase in the stability of its mRNA. Pilot studies indicate that the stability of transgenic human gamma-globin mRNA increases more than six-fold in mice homozygous for an unlinked beta-thalassemic determinant, and is correspondingly reduced in otherwise identical mice that transcribe human beta-globin mRNA from an independent, randomly-integrated transgene. In vitro analyses indicate that this effect may be mediated by conserved elements within the gamma- and beta-globin 3' UTRs that co-regulate the stability of the two mRNAs. Three experimental aims have been designed to confirm and extend these preliminary results. First, the importance of the dynamic regulation of gamma-globin mRNA stability during fetal and adult development will be established in transgenic mice using a novel assay designed specifically for this purpose. The effect of increased stability on the structure and translational efficiency of the gamma-globin mRNA will also be studied. Second, the likelihood that elements within the 3'UTR participate in regulating gamma-globin mRNA stability will be investigated by a series of linked structure-function studies. The stabilities of structurally-informative chimeric gamma-globin mRNAs will be determined in transgenic mice, and the results used to direct fine-mapping of crucial cis elements using both conventional in vitro methods and a newly-developed in vitro mRNA decay assay. Third, whole animal experiments will be carried out to test cell-culture and transgenic mouse evidence indicating that the stability of gamma-globin mRNA is enhanced by hydroxyurea and alpha-amino butyric acid, two gamma-globin inducing agents that have been used in human trials. Results from the proposed experiments will provide a more comprehensive understanding of the regulation of human gamma globin in both normal and pathological settings, as well as identify new mechanisms that can be targeted for therapeutic dysregulation in individuals with beta-globin gene disorders.

描述：（研究者的摘要）对 人类发育阶段限制性表达的分子基础 伽玛珠蛋白对于开发旨在重新激活其的方法至关重要 在患有先天性β链缺陷的个体中表达。虽然 胎儿到成人珠蛋白的转换主要是通过作用于的机制来实现的 基因转录水平，最近的研究表明 转录后事件也可能参与这一过程。目前的 该提案旨在调查生理和 γ-珠蛋白表达的药理学诱导可能与 其 mRNA 的稳定性增加。试点研究表明 转基因人伽马珠蛋白 mRNA 的稳定性增加了六倍以上 小鼠对于未连锁的β-地中海贫血决定子是纯合的，并且是 在转录人类的其他相同小鼠中相应减少 来自独立、随机整合的转基因的 β-珠蛋白 mRNA。体外 分析表明，这种效应可能是由内部保守元件介导的 共同调节两者稳定性的 γ 球蛋白和 β 球蛋白 3' UTR mRNA。设计了三个实验目标来确认和扩展这些 初步结果。一、动态调控的重要性 胎儿和成人发育期间的 γ-珠蛋白 mRNA 稳定性将 使用专门设计的新测定法在转基因小鼠中建立 这个目的。增加稳定性对结构的影响 γ-珠蛋白 mRNA 的翻译效率也将被研究。第二， 3'UTR 内的元件参与调节的可能性 γ-珠蛋白 mRNA 稳定性将通过一系列相关联的研究进行研究 结构-功能研究。结构信息的稳定性 将在转基因小鼠中测定嵌合γ-珠蛋白 mRNA，并且 结果用于使用两者直接精细映射关键的顺式元素 传统的体外方法和新开发的体外 mRNA 衰减测定。 第三，将进行整体动物实验来测试细胞培养和 转基因小鼠证据表明，γ-珠蛋白 mRNA 的稳定性 羟基脲和α-氨基丁酸增强，两种γ-珠蛋白诱导 已用于人体试验的药物。拟议的结果 实验将提供对调节更全面的理解 正常和病理环境下的人类伽马珠蛋白，以及 确定可针对治疗失调的新机制 患有β-珠蛋白基因疾病的个体。