Dynamic Regulation of y-Globin mRNA Stability

描述：(调查人员摘要)全面了解 人类发育阶段限制性表达的分子基础 伽马珠蛋白对于设计用来重新激活其 先天β链缺陷个体的表达。尽管 胎儿到成人的珠蛋白转换主要是通过作用于 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。海流 一项提案旨在调查生理学和 药物诱导γ-珠蛋白表达可能与一种 提高其mRNA的稳定性。初步研究显示， 转基因人丙种球蛋白基因的稳定性提高了6倍以上 纯合的未连接的β-地中海贫血决定簇，并且是 在转录人类基因的完全相同的小鼠中相应减少 来自独立的、随机整合的转基因的β-珠蛋白基因。离体 分析表明，这种效应可能是由体内的保守元件介导的 共同调节两者稳定性的γ-和β-珠蛋白3‘端非编码区 MRNAs。已经设计了三个实验目标来证实和扩展这些 初步结果。第一，动态监管的重要性 在胎儿和成人发育过程中，伽玛珠蛋白mRNA的稳定性 在转基因小鼠中建立了一种专门为 这就是目的。增加稳定性对结构和性能的影响 还将研究伽马珠蛋白mRNA的翻译效率。第二, 3‘非编码区内元件参与调控的可能性 将通过一系列连锁的研究来研究γ-珠蛋白mRNA的稳定性。 结构-功能研究。结构信息型的稳定性 将在转基因小鼠中确定嵌合的伽马珠蛋白mRNAs，并且 结果用于指导关键顺式元素的精细映射 常规的体外方法和新发展的体外mRNA衰变分析方法。 第三，将进行全动物实验，以测试细胞培养和 转基因小鼠证据表明，伽玛珠蛋白mRNA的稳定性是 羟基脲和α-氨基丁酸增强两种丙种球蛋白诱导 已经在人体试验中使用的药物。建议的结果 实验将提供对这一规定的更全面的理解 在正常和病理环境下的人丙种球蛋白，以及 找出可作为治疗失调症靶点的新机制 患有β-珠蛋白基因紊乱的个体。

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.