Protein RNA Rearrangements in the Spliceosome

描述（由申请人提供）：剪接体从前体信使RNA切除内含子是几乎所有人类基因表达的关键步骤。这一过程受到高度调控，与基因转录和其他加工事件（如聚腺苷酸化和核苷酸修饰）紧密相关。更好地了解前mRNA剪接将是至关重要的，以进一步了解机制，调节剪接，控制模式的选择性剪接，并有助于发展，肿瘤发生和逆转录病毒感染。 剪接体识别化学事件的确切位点的机制以及反应如何被催化还没有很好的理解。这个项目的长期目标是了解剪接体成分和RNA配体之间的相互作用和重排，RNA配体是催化反应的底物。有充分的证据表明，在分支位点上存在多个因子的重排和多个识别事件。这些事件的调查-这是不理解的机制-将阐明核心组件之间的相互作用和重排，并可能作为其他重排和多个识别事件发生在剪接体的其他地方的范例。该建议首先关注剪接体组装的第一个ATP依赖性步骤-分支区域周围U2 snRNP的稳定结合，并研究ATP酶Prp 5的作用。作为第一个ATP依赖性事件，这一步骤提供了一个独特的简化系统，用于研究剪接体ATP酶的作用。 进一步的实验将集中在机制和最近确定的桥接之间的相互作用的U1和U2 snRNP的Prp 5介导的后果。最后，使用一种新的遗传筛选，我们正在研究分支位点之间的相互作用（以及5'和3'剪接位点）和对第二催化反应至关重要的剪接体的组分，鉴定的组分和RNA底物之间的相互作用，以及所识别的成分与剪接体其他成分的相互作用--对于这些组分相互作用以帮助并列第二化学步骤的反应物的机理具有特定的弯曲。

DESCRIPTION (provided by applicant): Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how he reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events -- which are not understood mechanistically -- will elucidate interactions and rearrangements among core components and may serve as a paradigm for other rearrangements and multiple recognition events that occur elsewhere in the spliceosome. This proposal focuses first on the first ATP-dependent step of spliceosome assembly - the stable binding of U2 snRNP around the branch region, and investigates the action of an ATPase, Prp5. As the first ATP-dependent event, this step provides a uniquely simplified system, for studying the action of a spliceosomal ATPase. Further experiments will focus on the mechanism and the consequences of a recently identified bridging interaction between U1 and U2 snRNPs that is mediated by Prp5. Finally, using a new genetic screen, we are investigating interactions between the branch site (and the 5' and 3' splices sites) and components of the spliceosome critical for the second catalytic reaction, interactions between the identified components and the RNA substrate, and interactions of the identified components with other constituents of the spliceosome -- with a particular bent as to mechanism by which these components interact to help juxtapose the reactants for the second chemical step.