Regulation and Function of a Human Embryonic Globin

描述（由申请人提供）：该提案扩展了一项正在进行的研究计划，该计划致力于确定β-珠蛋白表达障碍成人中ε-珠蛋白基因再激活的潜在治疗价值。ε-珠蛋白在这些个体中的潜在益处来自于其掺入Hb异源四聚体中，这些异源四聚体显示生理学上适当的O2结合和抗镰状化特性。由于转录后机制在其他珠蛋白基因的调控表达中起着重要作用，因此影响ε-珠蛋白mRNA稳定性和翻译效率的类似过程可能对其表达同样重要。在完整的动物和体外进行的试点研究牵连特定的顺式序列和定义的反式作用因子参与调节ε-珠蛋白mRNA的稳定性，其中几个是高度相似的成熟阶段β-珠蛋白mRNA的稳定性的充分描述的决定因素。目前的建议将这些试点研究扩展到三个特定目的，调查调节ε-珠蛋白mRNA稳定性的独立方面。这些目标利用了利用申请人经验的成熟技术，以及几种在初步研究中得到验证的新方法。目的通过对ε-珠蛋白mRNA变异体的细胞培养分析，确定决定ε-珠蛋白mRNA稳定性的特异性顺式作用元件。这些决定因素的生理重要性随后将在完善的转基因小鼠模型系统中得到验证。目的II将确定影响ε-珠蛋白mRNA特征稳定性的特异性细胞质因子，并且还可能参与共调节β-和γ-珠蛋白mRNA的稳定性。这些实验将使用熟悉的分析方法在体外和体内进行。目的III将研究调节ε-珠蛋白mRNA稳定性对关键生理和分子过程的影响，包括其发育阶段限制的表达及其在定形红系细胞中的翻译效率。这些实验将利用申请人实验室为此特定目的开发的几种不寻常但信息量很大的方法。从所有三个目标的结果将整合，以提供一个坚实的理解的基本分子过程调节ε-珠蛋白mRNA的稳定性，这些过程可能会影响其他珠蛋白mRNA的稳定性的方式，以及生理后果-无论是可取的和不可取的-从他们的目标失调的结果。拟议的研究包括评估β地中海贫血和镰状细胞性贫血个体发育沉默珠蛋白基因的前景的关键一步。

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.