Nucleolin-mediated stabilization of human B-globin mRNA

核仁素介导的人 B 珠蛋白 mRNA 的稳定性

• 批准号: 7810541
• 负责人: J ERIC RUSSELL
• 金额: $ 39.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-06 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7810541
• 关键词: 3' Untranslated Regions; Address; Affinity; Be++ element; Beryllium; Binding; Binding Sites; Characteristics; Cultured Cells; Data; Elements; Erythroid Cells; Gene Expression; Globin; Goals; Human; In Situ; In Vitro; Laboratories; Length; Ligand Binding; Location; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular; Mutation; Nature; Participant; Play; Positioning Attribute; Post-Translational Protein Processing; Process; RNA Folding; RNA Recognition Motif; Reagent; Research; Role; Sickle Cell Anemia; Site; Specific qualifier value; Specificity; Structure; System; Thalassemia; To specify; Untranslated Regions; base; beta Globin; design; experience; in vivo; insight; interest; mRNA Stability; novel; nucleolin; research study; stem

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.

描述（由申请人提供）： 人类的正常表达？珠蛋白主要依赖于其编码 mRNA 异常高的稳定性。当前的提案旨在具体说明新描述的分子机制的基本特征，该分子机制似乎决定了人 α-珠蛋白 mRNA 在体内的组成稳定性。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原理。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，并证明该元件与核仁素（一种多功能、结构异质因子）反式结合。重要的是，消除核仁素结合的位点特异性突变也会破坏完整培养细胞中全长 β-珠蛋白 mRNA 原位的稳定性。 RNA 折叠模型预测 β-球蛋白 3'UTR 内存在强大的茎环结构，其中包含与 βCP 功能性结合位点直接相对的核仁素结合位点，βCP 是在调节 β-CP 稳定性中发挥关键作用的因子。 β-珠蛋白mRNA。基于这些数据，我们提出了一个β-珠蛋白 mRNA 稳定性模型，其中核仁素重塑 p-珠蛋白 3'UTR 内的二级结构，以促进 βCP 进入其功能结合位点。修订后的提案包含新的、确凿的研究，表明核仁素在体外显着增加了 ?CP 对天然 p-珠蛋白 3'UTR 的亲和力。新的研究还表明，核仁素的 β-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。目前的提案将初步研究扩展到三个目标，利用申请人在 mRNA 稳定性领域的长期兴趣和相关经验。目标 I 通过识别核仁素的参与 RNA 结合结构域并指定相关翻译后修饰的位置和性质，为核仁素的 β-球蛋白 mRNA 配体结合特异性建立结构基础。目的 II 验证了以下假设：β-球蛋白 3'UTR 内的天然二级结构禁止 βCP 与其功能结合位点结合，并直接解决了核仁素在重塑该区域中所发挥的作用。目标 III 定义了核仁素介导的 3'UTR 重塑的其他可能后果，包括分别对新生和成熟 β-珠蛋白 mRNA 的加工和翻译效率的影响。实验计划使用已在申请人实验室验证的试剂和方法，以及有助于成功完成既定目标的创造性方法。所提出的实验结果将为调节 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 β-珠蛋白 mRNA 稳定性的新疗法提供坚实的基础。地中海贫血和镰状细胞病调节β-珠蛋白基因表达的这一关键决定因素。