Spliceosomal snRNA modification in Xenopus oocytes

申请人描述：主要剪接体单链RNA U1、U2、U4、U5和U6 分享转录后修饰的共同特征，包括5‘帽 甲基化、内部假尿嘧啶和2‘-O-甲基化。修改后的 这些单链RNA中的核苷酸在不同物种之间非常保守， 强烈表明它们在生物学上具有重要意义。远景目标 这个项目的主要目的是了解 剪接体SnRNA修饰。对这一过程的详细调查已经 由于缺乏有效的分析和实验系统而受到阻碍。 最近，我们的实验室和其他实验室开发了几种高灵敏度的检测方法 检测RNA中的修饰核苷酸。利用非洲爪哇的卵母细胞，我们还开发了 一种用于执行非常高效的修改的实验系统 剪接体SnRNA。这两个发展第一次为我们提供了 解决剪接体SnRNA基本问题的工具 修改。因为它是经过最广泛修改的，所以U2是 我们的书房。这项提议有三个主要目标。(1)确定哪些 U2活性需要修饰的核苷酸，以及这些核苷酸是如何 为U2功能贡献力量。我们将生成各种U2构造，其中 介绍了选择性修饰或不可修饰的核苷酸。一种能力 这些U2衍生品中的一部分来重组U2 SnRNP组装并参与 剪接将被测试，重要的修饰核苷酸将被精确 下定决心。然后，这些关键的修饰核苷酸将被用于 4-Thiou位点特定的交联度分析。(2)解决亚蜂窝问题 负责U2内部改造的机械的国产化。我们 将使用系统的突变分析来确定关键的序列元素 U2是核苷酸修饰和适当的 通过荧光免疫细胞化学监测亚室靶向。 然后，缺少这些关键序列元件的突变U2 RNA将被连接 不同的信号序列，以靶向我们的实验U2 RNA 所需的亚细胞隔间。U2突变体的修饰修复 将提供关于U2内部修改发生在哪里的有价值的信息。 (3)明确参与U2修饰的细胞机制。我们的 初步结果表明，U2的2‘-O-甲基化是由核介导的 引导RNA，以类似于rRNA的方式。我们将使用各种 寻找和鉴定这种假定的引导RNA的生化方法 修饰活性负责2‘-O-甲基化和假尿嘧啶。

APPLICANT'S DESCRIPTION: The major spliceosomal snRNAs U1, U2, U4, U5, and U6 share common features of post-transcriptional modification, including 5' cap methylation and internal pseudouridylation and 2'-O-methylation. The modified nucleotides in these snRNAs are remarkably conserved from species to species, strongly suggesting that they are biologically significant. The long-range goal of this project is to understand the underlying biological principles of spliceosomal snRNA modifications. Detailed investigation of this process has been impeded by the lack of effective assays and experimental systems. Recently, our lab and others developed several highly sensitive assays for detecting modified nucleotides in RNA. Using Xenopus oocytes, we also developed an experimental system for performing extremely efficient modifications in spliceosomal snRNAs. For the first time, these two developments provide us with tools to address the fundamental questions regarding spliceosomal snRNA modification. Because it is the most extensively modified, U2 is the focus of our study. There are three major goals in this proposal. (1) To identify which modified nucleotides are required for U2 activity, and how these nucleotides contribute to U2 function. We will generate a variety of U2 constructs in which selectively modified or un-modifiable nucleotides are introduced. The ability of these U2 derivatives to reconstitute U2 snRNP assembly and participate in splicing will be tested and important modified nucleotides will be precisely determined. These critical modified nucleotides will then be targeted for 4-thioU site-specific cross-linking analysis. (2) To address the subcellular localization of the machinery responsible for U2 internal modifications. We will use a systematic mutational analysis to identify key sequence elements in U2 that are required for nucleotide modification and for proper subcompartmental targeting as monitored by fluorescence immunocytochemistry. Mutant U2 RNAs lacking these critical sequence elements will then be attached to various signal sequences in order to target our experimental U2 RNAs to desired subcellular compartments. Restoration of modification in U2 mutants will provide valuable information as to where U2 internal modifications occur. (3) To identify the cellular machinery involved in U2 modification. Our preliminary results suggest that 2'-O-methylation of U2 is mediated by nuclear guide RNAs, in a manner similar to that for rRNAs. We will use a variety of biochemical approaches to search for such putative guide RNAs and to identify the modifying activity responsible for 2'-O-methylation and pseudouridylation.