Nucleolin-mediated stabilization of human B-globin mRNA

性状（申请人提供）：人正常表达？珠蛋白严重依赖于其编码mRNA的异常高的稳定性。目前的建议是为了指定一个新描述的分子机制，似乎决定了人类的组成稳定性的基本特征？体内珠蛋白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。这些研究使用了一种新的原位方法来鉴定β-珠蛋白mRNA稳定性的一个以前未知的顺式决定簇，并证明该元件被核仁素（一种多功能、结构异质性因子）反式结合。重要的是，位点特异性突变，消融核仁素结合也不稳定的全长？珠蛋白mRNA在完整培养细胞中的原位表达。RNA折叠模型预测了？珠蛋白3 'UTR，包括核仁结合位点直接反对的功能结合位点？CP是一种在调节？-珠蛋白mRNA。根据这些数据，我们提出了一个模型？-珠蛋白mRNA的稳定性，其中核仁素重塑β-珠蛋白3 'UTR内的二级结构，以促进？CP进入其功能结合位点。修订后的建议包含新的，确证的研究表明，核仁素显着增加的亲和力？体外天然β-珠蛋白3 'UTR的CP。新的研究还表明，核仁素的？球蛋白mRNA配体结合特异性至少部分由其翻译后核糖基化提供。目前的提案扩展了三个目标的初步研究，利用了申请人在mRNA稳定性领域的长期兴趣和相关经验。目的一是建立一个结构基础？-通过鉴定核仁素的参与RNA结合结构域，并通过指定相关翻译后修饰的位置和性质，来确定核仁素的球蛋白mRNA配体结合特异性。目的二验证了假设，天然的二级结构内？-珠蛋白3 'UTR禁止？CP结合到其功能结合位点，并直接解决了核仁素在重塑该区域中所起的作用。目的III定义了核仁素介导的3 'UTR重塑的其他可能后果，包括对新生和成熟的？珠蛋白mRNA。该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所述目标的创造性方法。从拟议的实验结果将提供独特的见解的分子系统，调节？珠蛋白mRNA的稳定性，提供了一个强有力的基础，合理设计的新疗法？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.