Spliceosomal snRNA modification in Xenopus oocytes

描述(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的内部修饰的机制和功能。彻底了解这些修饰的性质将大大有助于我们了解基因调控(换句话说，蛋白质在细胞中的表达是如何调控的)。针对这一目标，我们提出了三个具体目标。(1)剪接体SnRNA修饰酶活性的鉴定与鉴定。利用亲和层析技术，从非洲爪哇卵母细胞核提取液中分离剪接体SnRNA特异性修饰酶(sNO/scaRNPs)。鉴于每个sno/scaRNP包含一组共同的蛋白质和一个唯一的引导RNA，我们将通过构建RNA文库来纯化RNA组分并进行鉴定。我们还将筛选酵母GST-ORF蛋白库，以寻找催化酵母剪接体SnRNA修饰的修饰活性(sno/scaRNPs和/或纯蛋白质酶)。通过系统的表征，可能会鉴定出一套完整的剪接体SnRNA特异性修饰活性，从而加深我们对剪接体SnRNA修饰机制的理解。(2)U2单链RNA修饰的功能分析。使用非洲爪哇卵母细胞，我们将首先解剖和分离对SnRNP组装重要的伪尿苷(在U2分支点识别区)和对前mRNA剪接重要的伪尿苷。然后，我们将使用SELEX进一步分析那些对剪接重要的伪尿苷，以详细了解它们在剪接过程中如何对分支站点识别做出贡献。我们还将进行缺失分析，以确定U2假尿苷在酵母中的重要性。与酵母U2相比，脊椎动物U2在分支位点识别区域多含有3个伪尿苷，因此我们将在酵母U2中引入额外的伪尿苷来模拟脊椎动物的U2修饰，并利用酵母ACT1-CUP1报告基因系统来测试是否有更多的伪尿苷能够耐受脊椎动物Pre-mRNAs中发现的多样化的分支位点序列。这一分析应该是对上述非洲爪哇卵母细胞SELEX分析的补充。(3)RNA引导的信使核糖核酸/前信使核糖核酸修饰的鉴定和鉴定。基本上，对信使核糖核酸/前信使核糖核酸的修饰一无所知。我们的初步实验表明，信使核糖核酸和前信使核糖核酸可能含有2‘-O-甲基化的核苷酸和假尿苷。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RNaseH裂解的方法，以及使用改进的寡核苷酸探针的杂交方法，来系统地表征并可能定量检测mRNA/Pre-mRNA中的2‘-O-甲基化和假尿酸。确定信使核糖核酸/前信使核糖核酸的修饰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.