Regulation and Function of a Human Embryonic Globin

描述(由申请人提供)：这项提案扩展了一项正在进行的研究计划，该计划致力于确定在患有β-珠蛋白表达障碍的成年人中重新激活epsilon-珠蛋白基因的潜在治疗价值。在这些个体中，epsilon-珠蛋白的潜在益处来自于它与Hb异构体的结合，Hb异构体显示出生理上合适的O2结合和抗病特性。由于转录后机制在调控其他珠蛋白基因的表达中起着至关重要的作用，因此影响epsilon-珠蛋白mRNA的稳定性和翻译效率的类似过程可能对其表达也同样重要。在完整动物和体外进行的初步研究涉及特定的顺式序列，并将反式作用因子定义为参与调节的epsilon-珠蛋白mRNA稳定性，其中几个与成熟阶段β-珠蛋白mRNA稳定性的决定因素高度相似。目前的提案在三个具体目标上扩展了这些先导研究，这些目的是研究受调控的epsilon-珠蛋白mRNA稳定性的独立方面。这些目标利用了成熟的技术，借鉴了申请者的经验，以及几种已在初步研究中得到验证的新方法。目的通过对含有特定位点3‘非编码区突变的epsilon-珠蛋白mRNA变体的新型细胞培养分析，定义决定epsilon-珠蛋白mRNA稳定性的特定顺式作用元件。这些决定因素的生理重要性随后将在建立良好的转基因小鼠模型系统中得到验证。目的II将确定影响表型珠蛋白mRNAs特征稳定性的细胞质因子，并可能参与共同调节β-和γ-珠蛋白mRNAs的稳定性。这些实验将在体外和体内使用熟悉的分析方法进行。目的研究调节的epsilon-珠蛋白mRNA稳定性对关键的生理和分子过程的影响，包括其在特定的红系细胞中的发育阶段限制性表达和翻译效率。这些实验将利用申请人实验室为这一特定目的开发的几种不寻常但信息量很大的方法。所有这三个目标的结果将结合在一起，以提供对调节epsilon-珠蛋白mRNAs稳定性的基本分子过程的坚实理解，这些过程可能影响其他珠蛋白mRNAs稳定性的方式，以及它们定向失调所导致的生理后果--无论是可取的还是不可取的。这项拟议的研究包括评估发育沉默的珠蛋白基因对患有β-地中海贫血和镰状细胞性贫血的个体的前景的关键一步。

DESCRIPTION (provided by applicant): This proposal extends an ongoing research program that is committed to defining the latent therapeutic value of epsilon-globin gene reactivation in adults with disorders in beta-globin expression. The potential benefit of epsilon-globin in these individuals derives from its incorporation into Hb heterotetramers that display physiologically appropriate O2-binding and antisickling properties. As post-transcriptional mechanisms play essential roles in the regulated expression of other globin genes, it is likely that similar processes affecting the stability and translational efficiency of epsilon-globin mRNA are equally important to its expression. Pilot studies carried out in intact animals and in vitro implicate specific cis sequences and defined trans-acting factors as participants in regulated epsilon-globin mRNA stability, several of which are highly similar to well-described determinants of adult-stage beta-globin mRNA stability. The current proposal extends these pilot studies in three Specific Aims that investigate independent aspects of regulated epsilon-globin mRNA stability. The Aims utilize well-established techniques that draw upon the applicant's experience, as well as several novel methods that have been validated in preliminary studies. Aim I will define specific cis-acting elements that dictate the stability of epsilon-globin mRNA, through novel cell culture analysis of epsilon-globin mRNA variants containing defined site-specific 3'UTR mutations. The physiological importance of these determinants will subsequently be validated in a well-established transgenic mouse model system. Aim II will identify the specific cytoplasmic factors that effect the characteristic stability of epsilon-globin mRNA and may also participate in co-regulating the stabilities of beta- and gamma-globin mRNAs. These experiments will be carried out in vitro and in vivo using familiar analytical methods. Aim III will investigate the effect of regulated epsilon-globin mRNA stability on crucial physiological and molecular processes, including its developmental stage-restricted expression and its translational efficiency in definitive erythroid cells. These experiments will capitalize on several unusual but highly informative methods that have been developed for this specific purpose in the applicant's laboratory. The results from all three Aims will integrate to provide a solid understanding of the fundamental molecular processes regulating the stability of epsilon-globin mRNA, the manner in which these processes may affect the stability of other globin mRNAs, and the physiological consequences--both desirable and undesirable--that result from their targeted dysregulation. The proposed research comprises a crucial step in evaluating the promise of developmentally silenced globin genes for individuals with beta-thalassemia and sickle cell anemia.