Spliceosomal snRNA modification in Xenopus oocytes

申请人描述：主要剪接体snRNAs U1、U2、U4、U 5和U6 具有转录后修饰的共同特征，包括5'帽 甲基化和内部假尿苷化和2 ′-O-甲基化。修改后的 这些snRNA中的核苷酸在物种之间是非常保守的， 强烈表明它们具有生物学意义。远景目标 这个项目的目的是了解潜在的生物学原理， 剪接体snRNA修饰。对这一过程的详细研究 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人开发了几种高度敏感的检测方法， 检测RNA中修饰的核苷酸。使用非洲爪蟾卵母细胞，我们还开发了 一个实验系统，用于执行非常有效的修改， 剪接体snRNAs。这两项发展首次为我们提供了 解决剪接体snRNA基本问题的工具 改性因为它是最广泛的修改，U2是重点， 我们的研究这项建议有三个主要目标。(1)来识别哪些 U2活性需要修饰的核苷酸，以及这些核苷酸如何 有助于U2功能。我们将生成各种U2结构，其中 引入选择性修饰的或不可修饰的核苷酸。的能力 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地 测定然后，这些关键的修饰的核苷酸将被靶向， 4-thioU位点特异性交联分析。(2)为了解决亚细胞 负责U2内部修改的机器的本地化。我们 将使用系统的突变分析来确定关键的序列元件， U2是核苷酸修饰和适当的 通过荧光免疫细胞化学监测的亚隔室靶向。 然后将缺乏这些关键序列元件的突变U2 RNA连接到 各种信号序列，以使我们的实验U2 RNA靶向 所需的亚细胞区室。U2突变体中修饰的恢复 将提供关于U2内部修改发生的有价值的信息。 (3)确定U2修饰所涉及的细胞机制。我们 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 生物化学方法来寻找这种推定的指导RNA，并确定 负责2 '-O-甲基化和假尿苷化的修饰活性。

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.