Spliceosomal snRNA modification in Xenopus oocytes

描述（由申请人提供）：我们研究的长期目标是详细了解剪接体snrna和mRNA/pre-mRNA内部修饰的机制和功能。彻底了解这些修饰的本质将大大有助于我们了解基因调控（换句话说，细胞中蛋白质表达是如何被调节的）。为此，我们提出三个具体目标。(1)剪接体snRNA修饰酶活性的鉴定和表征。利用亲和层析技术，从爪蟾卵母细胞核提取物中分离出剪接体snrna特异性修饰酶（sno/scaRNPs）。鉴于每个sno/scaRNP都含有一组共同的蛋白和一个独特的引导RNA，我们将纯化RNA组分，并通过构建RNA文库对其进行鉴定。我们还将筛选酵母GST-ORF蛋白文库，以寻找催化酵母剪接体snRNA修饰的修饰活性（sno/scaRNPs和/或蛋白酶）。通过系统的表征，将有可能鉴定出一整套剪接体snRNA特异性修饰活性，从而增强我们对剪接体snRNA修饰机制的认识。(2) U2 snRNA修饰的功能分析。使用非洲爪蟾卵母细胞，我们将首先解剖并解耦snRNP组装重要的假尿嘧啶（在U2分支位点识别区域）和pre-mRNA剪接重要的假尿嘧啶。然后，我们将使用SELEX进一步分析那些对剪接很重要的假嘧啶，以便详细了解它们如何在剪接过程中对分支位点识别做出贡献。我们还将进行缺失分析，以确定U2假尿嘧啶在酵母中的重要性。与酵母U2相比，脊椎动物U2在分支位点识别区多含有3个假尿嘧啶，因此我们将在酵母U2中引入额外的假尿嘧啶来模拟脊椎动物U2的修饰，并利用酵母ACT1-CUP1报告基因系统测试是否更多的假尿嘧啶可以耐受脊椎动物pre- mrna中发现的多种分支位点序列。该分析应补充上述非洲爪蟾卵母细胞SELEX分析。(3) rna引导的mRNA/pre-mRNA修饰的鉴定和表征。基本上我们对mRNA/pre-mRNA的修饰一无所知。我们的初步实验表明mRNA和pre-mRNA含有2'- o -甲基化核苷酸和假尿嘧啶。我们将使用现有的敏感方法，包括基于核酸酶tlc、引物延伸、位点特异性RNase H切割和使用修饰的寡核苷酸探针杂交的方法，系统地表征和可能量化mRNA/pre-mRNA中的2'- o甲基化和假尿嘧啶化。mRNA/pre- mRNA修饰的鉴定将成为未来功能分析的基础。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand, in detail, the mechanism and function of internal modifications of spliceosomal snRNAs and mRNA/pre-mRNA. A thorough understanding of the nature of these modifications will contribute greatly to our knowledge of gene regulation (in other words, how protein expression is regulated in the cell). Toward this goal, we propose three specific aims. (1) Identification and characterization of enzymatic activities for spliceosomal snRNA modifications. Using affinity chromatography, we will isolate spliceosomal snRNA-specific modifying enzymes (sno/scaRNPs) from Xenopus oocyte nuclear extracts. Given that each sno/scaRNP contains a set of common proteins and a unique guide RNA, we will purify the RNA components and identify them through the construction of an RNA library. We will also screen the yeast GST-ORF protein library for modifying activities (sno/scaRNPs and/or protein-only enzymes) catalyzing yeast spliceosomal snRNA modifications. Through systematic characterization, a complete set of spliceosomal snRNA-specific modifying activities will likely be identified, thus enhancing our understanding of the mechanism of spliceosomal snRNA modifications. (2) Functional analysis of U2 snRNA modifications. Using Xenopus oocytes, we will first dissect and uncouple the pseudouridines (in the U2 branch site recognition region) important for snRNP assembly from those important for pre-mRNA splicing. We will then further analyze those pseudouridines important for splicing using SELEX to allow a detailed understanding of how they contribute to the branch site recognition during splicing. We will also carry out deletional analysis to establish the importance of U2 pseudouridines in yeast. Vertebrate U2 contains three more pseudouridines in the branch site recognition region compared with yeast U2, and thus we will introduce extra pseudouridines into yeast U2 to mimic the vertebrate U2 modifications and test, using the yeast ACT1-CUP1 reporter gene system, whether more pseudouridines can tolerate the diverse branch site sequences found in vertebrate pre-mRNAs. This analysis should complement the above SELEX analysis in Xenopus oocytes. (3) Identification and characterization of RNA-guided mRNA/pre-mRNA modifications. Essentially nothing is known about mRNA/pre-mRNA modifications. Our preliminary experiments suggest that mRNA and perhaps pre-mRNA contain 2'-O-methylated nucleotides and pseudouridines. We will use the available sensitive methods, including those based on nuclease-TLC, primer-extension, site-specific RNase H cleavage, and hybridization using modified oligonucleotide probes, to systematically characterize and possibly quantify 2'-O-methylation and pseudouridylation in mRNA/pre-mRNA. Identification of mRNA/pre- mRNA modifications will form the basis for future functional analyses.