MECHANISMS FOR REARRANGING RNA DURING PRE-MRNA SPLICING

mRNA 前剪接过程中 RNA 重排机制

• 批准号: 6636541
• 负责人: JONATHAN P STALEY
• 金额: $ 20.39万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636541
• 关键词: RNA splicing; Saccharomyces cerevisiae; adenosinetriphosphatase; enzyme substrate; fungal genetics; precursor mRNA; spliceosomes

DESCRIPTION (from the application): Rearrangement of both RNA and DNA structure is critical to many basic biological processes, including pre-mRNA splicing. Upon interaction with a pre-mRNA substrate, the spliceosome undergoes a series of dramatic RNA rearrangements that configure the spliceosome for catalysis. Strikingly, these rearrangements reflect the disruption and formation of mutually exclusive structures. This mutually excusive design is a hallmark of splicing and likely ensures that the spliceosome assembles in a highly coordinated and carefully regulated fashion. Although these rearrangements play a critical role in governing spliceosome activation, it is not clear how these rearrangements are catalyzed. RNA-stimulated ATPases of the ubiquitous DEAD-box family have been implicated in orchestrating these RNA rearrangements, but no target, either RNA or protein, has been proved. Moreover, to establish specificity and prevent spurious RNA rearrangements, the activity of these DEAD-box proteins must be tightly controlled, but mechanisms for regulating these ATPases have not yet been identified. Finally, the RNA folding pathway for the spliceosome likely reflects a hierarchy of RNA rearrangements carefully designed to regulate spliceosome activation, but this hierarchy remains to be defined. The long-term goal of this proposal is to address these issues by focusing on a critical rearrangement required for 5' splice site recognition, the switch of U1 for U6. A major aim is to determine how Prp28, a DEAD-box ATPase, catalyzes the switch. A second aim is to determine how the switch of U1 for U6 triggers unwinding of the U4/U6 duplex, a key step in the catalytic activation of the spliceosome. A final aim is to establish the hierarchy between the switch of U1 for U6 and unwinding of the 5' stem-loop of U2, another critical rearrangement required to catalytically activate the spliceosome. To pursue these aims, a combined approach of genetics and biochemistry will be employed in the yeast S. cerevisiae. These studies will likely have broad implications for understanding (i) the function of DEAD-box proteins in their biologically relevant contexts, (ii) the mechanisms for regulating the timing and specificity of these ATPases, and (iii) the rationale underlying the dynamic design of the spliceosome. Since coupling of spliceosome activation with substrate recognition likely serves to ensure fidelity in splicing, these studies should also contribute insights into the mechanisms for preventing splicing errors that lead to human disorders, such as cancer and heart disease.

描述（来自应用程序）：RNA和DNA结构的重排 对于许多基本的生物过程至关重要，包括前MRNA剪接。 与前MRNA底物相互作用后，剪接体经历一个系列 戏剧性的RNA重排，以配置剪接体进行催化。 令人惊讶的是，这些重排反映了 相互排斥的结构。这种互惠性设计是 拼接并可能确保剪接体在高度中组装 进行了协调和精心监管的时尚。虽然这些重新排列 在管理剪接体激活中的关键作用，尚不清楚这些 重排是催化的。无处不在的死盒的RNA刺激的ATPase 一家人牵涉到编排这些RNA重排，但没有 靶标的RNA或蛋白质已被证明。此外，要建立 特异性并防止虚假的RNA重排，这些活性 死盒蛋白必须受到严格控制，但是调节的机制 这些ATPases尚未确定。最后，RNA折叠途径 因为剪接可能会仔细反映RNA重排的层次结构 设计用于调节剪接体激活，但该层次结构仍然是 定义。该提议的长期目标是通过 专注于5'剪接站点识别所需的关键重排， U1的U1开关。 一个主要目的是确定Dead-Box ATPase PRP28如何催化开关。 第二个目的是确定U6触发器的U1转换如何放松 U4/U6双链体，这是剪接体催化激活的关键步骤。一个 最终目的是在U1和U1的开关之间建立层次结构 放松U2的5'茎环，这是另一个重要的重排 催化激活剪接体。追求这些目标，一个合并 遗传学和生物化学方法将采用酵母。 酿酒酵母。这些研究可能对理解具有广泛的影响 （i）死盒蛋白在其生物学相关的上下文中的功能， （ii）调节这些ATPases的时间和特异性的机制， （iii）剪接体动态设计的基本原理。自从 剪接体激活与底物识别的耦合可能有助于 确保在剪接方面保真，这些研究也应促进见解 防止剪接错误导致人类疾病的机制， 例如癌症和心脏病。