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性质将大大有助于我们了解基因调控（换句话说，蛋白质表达如何在细胞中调节）。为了实现这一目标，我们提出了三个具体目标。(1)剪接体snRNA修饰的酶活性的鉴定和表征。使用亲和层析，我们将分离剪接体的snRNA特异性修饰酶（sno/scaRNP）从爪蟾卵母细胞核提取物。鉴于每个sno/scaRNP包含一组共同的蛋白质和一个独特的向导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蛋白文库中催化酵母剪接体snRNA修饰的修饰活性（sno/scaRNP和/或仅蛋白酶）。通过系统的表征，一套完整的spliceosomal snRNA特异性修饰活动将有可能被确定，从而提高我们对spliceosomal snRNA修饰机制的理解。(2)U2 snRNA修饰的功能分析。使用非洲爪蟾卵母细胞，我们将首先解剖和解偶联的pseudouridines（在U2分支网站识别区）重要的snRNP大会从那些重要的前mRNA剪接。然后，我们将进一步分析那些假尿苷重要的剪接使用SELEX，让他们如何有助于剪接过程中的分支位点识别的详细了解。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在分支位点识别区中含有多三个假尿苷，因此我们将额外的假尿苷引入酵母U2中以模拟脊椎动物U2的修饰，并使用酵母ACT 1-CUP 1报告基因系统测试是否有更多的假尿苷可以耐受在脊椎动物前mRNA中发现的多样性分支位点序列。该分析应补充上述在非洲爪蟾卵母细胞中的SELEX分析。(3)RNA引导的mRNA/前体mRNA修饰的鉴定和表征。基本上对mRNA/前mRNA修饰一无所知。我们的初步实验表明，mRNA和可能的前mRNA含有2 '-O-甲基化的核苷酸和假尿苷。我们将使用现有的灵敏方法，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的杂交的方法，系统地表征和可能定量mRNA/前mRNA中的2 '-O-甲基化和假尿苷化。mRNA/前mRNA修饰的鉴定将形成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.