Nucleolin-mediated stabilization of human B-globin mRNA

描述(申请人提供)：人类的正常表情？珠蛋白在很大程度上依赖于其编码信使核糖核酸的高度稳定性。目前的提议旨在明确一种新描述的分子机制的基本特征，该机制似乎决定了活体中人β-珠蛋白mRNA的构成稳定性。九项初步研究为本申请中详述的研究目标提供了令人信服的科学依据。这些研究使用一种新的原位方法来鉴定一个以前未知的p-珠蛋白mRNA稳定性的顺式决定因素，并证明该元件与核仁反式结合，核仁是一种多功能的结构不同的因子。重要的是，破坏核仁素结合的定点突变也破坏了完整培养细胞中全长？-珠蛋白mRNA的原位稳定。RNA折叠模型预测了β-珠蛋白3‘非编码区内有一个强大的茎环结构，包括核仁素结合部位，与？CP的功能结合部位直接相对，这是一个在调节β-珠蛋白mRNA稳定性方面起关键作用的因素。基于这一数据，我们提出了一个关于β-珠蛋白mRNA稳定性的模型，在该模型中，核仁会重塑p-珠蛋白3‘非编码区的二级结构，以促进？CP进入其功能结合部位。修订后的提案包含了新的确证研究，证明核素在体外显著增加了？CP与天然p-珠蛋白3‘非编码区的亲和力。新的研究还表明，核素的？-珠蛋白mRNA配体结合的特异性，至少部分是由其翻译后核糖化提供的。目前的建议将初步研究扩展为三个目标，这三个目标利用了申请人在信使核糖核酸稳定性领域的长期兴趣和相关经验。目的I通过鉴定其参与的RNA结合域，以及相关的翻译后修饰的位置和性质，为核仁β-珠蛋白mRNA配体结合的特异性奠定了结构基础。目的II证实了β-珠蛋白3‘非编码区的天然二级结构阻止？CP与其功能结合部位结合的假说，并直接阐述了核素在该区域重塑中所起的作用。目的III确定核仁素介导的3‘非编码区重塑的其他可能后果，包括分别对新生和成熟的珠蛋白mRNAs的加工和翻译效率的影响。实验计划利用了在申请人的实验室中经过验证的试剂和方法，以及有助于成功完成所述目标的创造性方法。这些实验的结果将为调节β-珠蛋白mRNA稳定性的分子系统提供独特的见解，为合理设计新的治疗方法提供强有力的依据。地中海贫血和镰状细胞疾病，调节这个关键的决定因子-珠蛋白基因的表达。

DESCRIPTION (provided by applicant): The normal expression of human ? globin is critically dependent upon the unusually high stability of its encoding mRNA. The current proposal is designed to specify the fundamental features of a newly described molecular mechanism that appears to dictate the constitutive stability of human ?-globin mRNA in vivo. Nine Preliminary Studies provide compelling scientific rationale for the research aims detailed in this application. These studies use a novel in situ method to identify a previously unknown cis-determinant of p-globin mRNA stability, and demonstrate that this element is bound in trans by nucleolin, a multi- functional, structurally heterogeneous factor. Importantly, site-specific mutations that ablate nucleolin binding also destabilize the full-length ?-globin mRNA in situ in intact cultured cells. RNA-folding models predict a strong stem-loop structure within the ?-globin 3'UTR, encompassing the nucleolin-binding site in direct opposition to a functional binding site for ?CP, a factor that plays a critical role in regulating the stability of ?-globin mRNA. Based upon this data, we propose a model for ?-globin mRNA stability in which nucleolin remodels secondary structure within the p-globin 3'UTR to facilitate ?CP access to its functional binding site. The revised proposal contains new, corroborating studies demonstrating that nucleolin dramatically increases the affinity of ?CP for the native p-globin 3'UTR in vitro. New studies also demonstrate that nucleolin's ?-globin mRNA ligand-binding specificity is provided, at least in part, by its post-translational ribosylation. The current proposal extends the Preliminary Studies in three Aims that capitalize on the applicant's longstanding interest and relevant experience in the field of mRNA stability. Aim I establishes a structural basis for the ?-globin mRNA ligand-binding specificity of nucleolin by identifying its participant RNA-binding domains, and by specifying the position and nature of relevant post-translational modifications. Aim II validates the hypothesis that native secondary structure within the ?-globin 3'UTR prohibits ?CP binding to its functional binding site, and directly addresses the proposed role that nucleolin plays in remodeling this region. Aim III defines other likely consequences of nucleolin-mediated 3'UTR remodeling, including effects on the processing and translational efficiency of nascent and mature ?-globin mRNAs, respectively. The Experimental Plan utilizes reagents and methods that have been validated in the applicant's laboratory, as well as creative approaches that will facilitate successful completion of the stated Aims. Results from the proposed experiments will afford unique insights into the molecular system that regulates ?-globin mRNA stability, providing a strong basis for the rational design of novel therapies for ? thalassemia and sickle cell disease that modulate this critical determinant of ?-globin gene expression.