Dynamic Regulation of y-Globin mRNA Stability

γ-珠蛋白 mRNA 稳定性的动态调节

• 批准号: 6650869
• 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/6650869
• 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 人类发育阶段限制表达的分子碱基 γ-球蛋白对于旨在重新激活其重新激活的方法的产生至关重要 先天性β链缺陷的个体的表达。虽然 胎儿到成年的环球蛋白开关主要是通过作用的机制来实现的 基因转录水平，最近的研究表明 转录后事件也可能参与此过程。电流 建议旨在调查生理和 γ-球蛋白表达的药理诱导可能与 提高其mRNA的稳定性。试点研究表明 转基因人γ--珠蛋白mRNA的稳定性增加了六倍以上 无链接β-盐杂质决定因素的纯合子的小鼠，IS 在抄写人类的原本相同的小鼠中相应减少了 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，这种效应可能是由内部的保守元素介导的 共同调节两者的稳定性的γ-和β-珠蛋白3' mrnas。已经设计了三个实验目标来确认和扩展这些 初步结果。首先，动态调节的重要性 胎儿和成人发育过程中的γ-球蛋白mRNA稳定性将是 使用专门设计的新颖测定法在转基因小鼠中建立 这个目的。稳定性提高对结构的影响 还将研究γ-球蛋白mRNA的翻译效率。第二， 3'utr中的要素参与调节的可能性 γ-球蛋白mRNA稳定性将通过一系列链接研究 结构功能研究。结构信息的稳定性 嵌合γ-球蛋白mRNA将在转基因小鼠中确定，并确定 结果用于指导至关重要的顺式元素使用两者 常规的体外方法和新开发的体外mRNA衰变测定法。 第三，将进行整个动物实验以测试细胞培养和 转基因小鼠的证据表明γ-球蛋白mRNA的稳定性是 羟基脲和α-氨基丁酸增强 已在人类试验中使用的代理商。提议的结果 实验将对法规提供更全面的理解 在正常和病理环境中，人类伽马球蛋白的 确定可以针对治疗失调的新机制 患有β-珠蛋白基因疾病的人。