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（来自申请）：RNA 和 DNA 结构的重排 对许多基本生物过程至关重要，包括前 mRNA 剪接。 在与前 mRNA 底物相互作用后，剪接体经历一系列 戏剧性的 RNA 重排，配置剪接体进行催化。 引人注目的是，这些重新安排反映了 互斥的结构。这种相互排斥的设计是 剪接并可能确保剪接体以高度组装 协调和精心调节的时尚。虽然这些重新安排发挥了作用 在控制剪接体激活中发挥关键作用，但尚不清楚这些如何 重排被催化。无处不在的 DEAD-box 的 RNA 刺激的 ATP 酶 家族参与了这些 RNA 重排的策划，但没有 目标，无论是RNA还是蛋白质，都已被证明。此外，要建立 特异性并防止虚假 RNA 重排，这些活性 死盒蛋白必须受到严格控制，但调节机制 这些ATP酶尚未被鉴定。最后，RNA折叠途径 因为剪接体可能仔细地反映了 RNA 重排的层次结构 旨在调节剪接体激活，但这种层次结构仍然是 定义的。该提案的长期目标是通过以下方式解决这些问题 专注于 5' 剪接位点识别所需的关键重排， 将U1切换到U6。 主要目的是确定 Prp28（一种死盒 ATP 酶）如何催化这种转换。 第二个目标是确定 U1 到 U6 的切换如何触发 U4/U6 双链体，剪接体催化激活的关键步骤。一个 最终目标是在 U1 到 U6 的切换之间建立层次结构 U2 5'茎环的解开，这是另一个关键的重排 催化激活剪接体。为了实现这些目标，联合 遗传学和生物化学的方法将被用于酵母S. 酿酒酵母。这些研究可能会对理解产生广泛的影响 (i) DEAD-box 蛋白在其生物学相关环境中的功能， (ii) 调节这些 ATP 酶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 确保剪接的保真度，这些研究还应有助于深入了解 防止导致人类疾病的剪接错误的机制， 例如癌症和心脏病。