Nucleolin-mediated stabilization of human B-globin mRNA

描述（申请人提供）：人的正常表达？珠蛋白在很大程度上依赖于其编码mRNA的异常高稳定性。目前的提案旨在详细说明一种新描述的分子机制的基本特征，这种机制似乎决定了人类的结构稳定性。-珠蛋白mRNA。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颖的原位方法来鉴定先前未知的p-珠蛋白mRNA稳定性的顺式决定因素，并证明该元件在反式中由核蛋白结合，核蛋白是一种多功能，结构异质因子。重要的是，位点特异性突变会破坏核蛋白结合，也会破坏全长？-珠蛋白mRNA在完整培养细胞中的原位表达。rna折叠模型预测在？-球蛋白3'UTR，包含核蛋白结合位点，与？CP，一个在调节？球蛋白mRNA。基于这些数据，我们提出了一个？-珠蛋白mRNA的稳定性，核蛋白在p-珠蛋白3'UTR内重塑二级结构以促进？CP进入其功能结合位点。修订后的提案包含新的、确凿的研究，表明核蛋白显著增加？体外天然p-珠蛋白3'UTR的CP。新的研究还表明，核蛋白的？-珠蛋白mRNA配体结合特异性至少部分是由其翻译后核糖基化提供的。目前的提案扩展了初步研究的三个目标，利用申请人在mRNA稳定性领域的长期兴趣和相关经验。目标1为？-globin mRNA配体结合特异性通过识别其参与rna结合域，并通过指定相关翻译后修饰的位置和性质。目的II验证了原生二级结构在？-globin 3'UTR禁止？CP结合到其功能结合位点，并直接解决了核蛋白在该区域重塑中所起的作用。Aim III定义了核蛋白介导的3'UTR重塑的其他可能后果，包括对新生和成熟细胞的加工和翻译效率的影响。-珠蛋白mrna。实验计划使用的试剂和方法已在申请人的实验室验证，以及创造性的方法，将有助于成功完成所述的目标。拟议实验的结果将为调控？-珠蛋白mRNA的稳定性，为合理设计新的治疗方法提供了强有力的基础。地中海贫血和镰状细胞病调节这一关键决定因素？-珠蛋白基因表达。

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.