Dynamic Regulation of y-Globin mRNA Stability

描述：（研究者摘要）全面了解 发育阶段限制性表达人 γ-珠蛋白是至关重要的方法，旨在重新激活它的一代。 在患有先天性β链缺陷的个体中表达。虽然 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， 转录后事件也可参与该过程。当前 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 增加其mRNA的稳定性。试点研究表明， 转基因人γ-珠蛋白mRNA的稳定性增加超过6倍， 对于非连锁β-地中海贫血决定簇纯合的小鼠， 在其他方面相同的小鼠中相应地减少， 来自独立的随机整合的转基因的β-珠蛋白mRNA。体外 分析表明，这种效应可能是由保守的元件， γ-和β-珠蛋白3'UTR共同调节两者的稳定性， mRNA。设计了三个实验目标来证实和扩展这些 初步结果。一、动态监管的重要性 胎儿和成人发育过程中γ-珠蛋白mRNA的稳定性将是 在转基因小鼠中建立，使用专门设计的新测定法， 这个目的。增加稳定性对结构的影响， 还将研究γ-珠蛋白mRNA的翻译效率。第二、 3 'UTR内的元件参与调节 γ-珠蛋白mRNA的稳定性将通过一系列连接的 结构-功能研究结构信息的稳定性 嵌合γ-珠蛋白mRNA将在转基因小鼠中测定， 结果用于使用两者指导关键顺式元件的精细映射 常规体外方法和新开发的体外mRNA衰变测定。 第三，将进行整体动物实验以测试细胞培养和 转基因小鼠的证据表明，γ-珠蛋白mRNA的稳定性， 由羟基脲和α-氨基丁酸增强，两种γ-珠蛋白诱导 已经在人体试验中使用的药物。拟议的结果 实验将提供更全面的了解该法规 人类γ珠蛋白在正常和病理情况下，以及 确定新的机制，可以针对治疗失调， 患有β-珠蛋白基因疾病的个体。

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.