EXON LIGATION--THE SPLICEOSOME AND GROUP 11 INTRONS

外显子连接——剪接体和第11组内含子

• 批准号: 2459644
• 负责人: Melissa J. Moore
• 金额: $ 18.26万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459644
• 关键词: RNA; RNA splicing; active sites; affinity labeling; chemical cleavage; chemical kinetics; crosslink; divalent cations; enzyme mechanism; enzyme substrate; esterification; introns; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; polynucleotides; precursor mRNA; pyrimidine nucleotides; ribozymes; small nuclear RNA; spliceosomes; thermodynamics; thiophosphate

The removal of introns by RNA splicing is an essential step in the expression of almost all human genes. Many nascent transcripts are subject to alternative splicing, which provides a means for making more than one protein from a single gene. Such alternative splicing can be either developmentally or tissue-specifically controlled. Whether or not an RNA is spliced is also subject to regulation: controlling the level of spliced and unspliced viral RNA in the cytoplasm is critical to the replication of HIV. Thus a detailed knowledge of the splicing process will be essential for better understanding of the basic mechanisms of gene expression, as well as organismal development, oncogenesis and the progression of retroviral infection. Because spliceosomal processing and the self-catalyzed excision of group II introns occur via the same two-step pathway, the two intron types are thought to be evolutionarily related. Therefore, the spliceosome has long been suspected to be an RNA catalyst. However, the precise makeup of the catalytic center(s) has remained elusive, primarily because spliceosomal components that interact directly with the splice site phosphates have yet to be identified. Neither is it known to what extent the active sites for the two steps of splicing overlap, although growing evidence supports some sort of structural rearrangement between the two steps. Even less is known about the catalytic mechanisms and active site structure of group II introns. Thus, whether the common two-step pathway has resulted from divergent or convergent evolution of these intron types remains debatable. Additionally, what mechanistic similarities exist, if any, between the above introns and introns of the group l self-splicing class are totally unknown. A long-term goal of this laboratory is to elucidate the catalytic mechanisms and active site machinery utilized by the spliceosome and group II introns to catalyze intron excision. This proposal focuses primarily on the exon ligation step. Parallel approaches to be used in both systems include: (l) development of a comprehensive kinetic framework for exon ligation using an assay in which the 3' splice site is added in trans; (2) use of chemically modified 3' splice site RNAs to determine the exact substrate structural requirements for exon ligation, and how these structures contribute to splice site recognition and catalysis; and (3) incorporation of photo- and affinity-crosslinking reagents to identify active site components closely juxtaposed to the phosphodiester backbone at the 3' splice site. Together, these experiments should allow direct comparison of active site structure, 3' splice site recognition and mechanisms of catalysis by the spliceosome and both group II and group I self-splicing introns.

通过RNA剪接去除内含子是RNA合成中的重要步骤。 几乎所有人类基因的表达。许多新生的转录本 进行选择性剪接，这提供了一种手段，使更多的 而不是一个基因的蛋白质。这种选择性剪接可以是 无论是发育还是组织特异性控制。是否 RNA的剪接也受到调控：控制水平 细胞质中剪接和未剪接的病毒RNA的相互作用对于 艾滋病毒的复制。因此，详细了解拼接过程 对于更好地理解生物学的基本机制至关重要。 基因表达，以及生物体发育，肿瘤发生和 逆转录病毒感染的进展。 因为剪接体加工和自催化的基团切除 II内含子通过相同的两步途径发生，这两种内含子类型是 被认为与进化有关。因此，剪接体长期以来 被怀疑是RNA催化剂。然而， 催化中心仍然难以捉摸，主要是因为剪接体 与剪接位点磷酸直接相互作用的组分具有 还有待确认也不知道活性位点在多大程度上 对于拼接重叠的两个步骤，尽管越来越多的证据支持 两个步骤之间的某种结构重排。更不 已知的催化机制和活性中心结构， II组内含子。因此，共同的两步途径是否导致了 从这些内含子类型的分歧或趋同进化仍然存在 值得商榷此外，如果存在机械相似性， 在上述内含子和组L自剪接类的内含子之间 是完全未知的。 这个实验室的一个长期目标是阐明 剪接体利用的机制和活性位点机制， II组内含子催化内含子切除。该提案重点 主要在外显子连接步骤上。 拟采用的并行办法 这两个系统包括：（l）发展一个全面的动力学 使用其中3'剪接位点被插入外显子连接的测定法， （2）使用化学修饰的3'剪接位点RNA， 确定外显子连接的确切底物结构要求， 以及这些结构如何有助于剪接位点识别， 催化;和（3）光交联和亲和交联的结合 试剂，以识别活性位点组分紧密并列的 在3'剪接位点的磷酸二酯骨架。所有这些 实验应允许直接比较活性位点结构，3' 剪接体对剪接位点的识别及其催化机制 以及II组和I组自剪接内含子。