Spliceosomal snRNA modification in Xenopus oocytes

非洲爪蟾卵母细胞中的剪接体 snRNA 修饰

• 批准号: 7225601
• 负责人: YI-TAO YU
• 金额: $ 27.27万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7225601
• 关键词: 5 fluorouridine; Address; Affinity Chromatography; Binding; Biochemical Genetics; Biological Assay; Boxing; Cells; Classification; Complement; Coupled; Data; Enzymes; Facility Construction Funding Category; Figs - dietary; Future; Gene Expression Regulation; Genomic Library; Glutathione S-Transferase; Goals; Guide RNA; Heterogeneous Nuclear RNA; Knowledge; Laboratories; Libraries; Maps; Messenger RNA; Methods; Methylation; Modification; Nature; Nuclear Extract; Nucleotides; Numbers; Oligonucleotide Probes; Open Reading Frames; Play; Point Mutation; Positioning Attribute; Primer Extension; Progress Reports; Proteins; Pseudouridine; RNA; RNA Splicing; RNA library; Radiolabeled; Reporter Genes; Reporting; Research; Research Personnel; Ribonuclease H; Ribosomal RNA; Role; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; System; Testing; Transfer RNA; U2 small nuclear RNA; Xenopus; Xenopus oocyte; Yeasts; base; cell growth; inhibitor/antagonist; mRNA Precursor; nuclease; programs; protein expression; radiotracer; reconstitution; research study; snRNP Biogenesis; yeast genetics; 5 fluorouridine; Address; Affinity Chromatography; Binding; Biochemical Genetics; Biological Assay; Boxing; Cells; Classification; Complement; Coupled; Data; Enzymes; Facility Construction Funding Category; Figs - dietary; Future; Gene Expression Regulation; Genomic Library; Glutathione S-Transferase; Goals; Guide RNA; Heterogeneous Nuclear RNA; Knowledge; Laboratories; Libraries; Maps; Messenger RNA; Methods; Methylation; Modification; Nature; Nuclear Extract; Nucleotides; Numbers; Oligonucleotide Probes; Open Reading Frames; Play; Point Mutation; Positioning Attribute; Primer Extension; Progress Reports; Proteins; Pseudouridine; RNA; RNA Splicing; RNA library; Radiolabeled; Reporter Genes; Reporting; Research; Research Personnel; Ribonuclease H; Ribosomal RNA; Role; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; System; Testing; Transfer RNA; U2 small nuclear RNA; Xenopus; Xenopus oocyte; Yeasts; base; cell growth; inhibitor/antagonist; mRNA Precursor; nuclease; programs; protein expression; radiotracer; reconstitution; research study; snRNP Biogenesis; yeast genetics

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.

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/mRNA/PRE-MRNA的内部修饰的机制和功能。对这些修饰的性质的透彻理解将极大地有助于我们对基因调节的了解（换句话说，如何调节细胞中蛋白质的表达）。为了实现这一目标，我们提出了三个具体目标。 （1）鉴定和表征用于剪接SNRNA修饰的酶活性。使用亲和色谱法，我们将从异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都包含一组常见蛋白和独特的指南RNA，我们将纯化RNA成分并通过构建RNA库来识别它们。我们还将筛选酵母GST-ORF蛋白质文库，以修饰活性（SNO/SCRNPS和/或仅蛋白质酶）催化酵母菌剪接体SNRNA修饰。通过系统的表征，可能会确定一组完整的剪接SNRNA特异性修饰活动，从而增强我们对剪接体SNRNA修饰机制的理解。 （2）U2 snRNA修饰的功能分析。使用爪蟾卵母细胞，我们将首先解剖并解开对SNRNP组装重要的伪基（在U2分支位点识别区域），从对前MRNA剪接的重要的组件中很重要。然后，我们将进一步分析那些对于使用SELEX拼接重要的伪源，以详细了解它们在拼接过程中如何贡献分支站点识别。我们还将进行缺失分析，以确定酵母中U2伪苷的重要性。 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.该分析应补充上述Xenopus卵母细胞中的SELEX分析。 （3）RNA引导的mRNA/前MRNA修饰的鉴定和表征。基本上，关于mRNA/前mRNA修饰的知之甚少。我们的初步实验表明mRNA和可能的mRNA包含2'-O-甲基化的核苷酸和伪苷。我们将使用可用的敏感方法，包括基于Nuclease-TLC，引物延伸，特异性RNase H裂解和使用改良的寡核苷酸探针进行杂交的方法，以系统地表征2'-O-O-甲基化和MRNA/Prena prena/pre-MRNA中的2'-O-甲基化和伪基化。 mRNA/PRE-mRNA修饰的识别将构成未来功能分析的基础。