RNA Splicing in Archaea

古细菌中的 RNA 剪接

• 批准号: 7910986
• 负责人: Ramesh Gupta
• 金额: $ 11.81万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910986
• 关键词: 2' -O-methyluridine; Academic Research Enhancement Awards; Animal Model; Animals; Archaea; Award; Boxing; Codon Nucleotides; Complex; Core Protein; Development; Differentiation and Growth; Enzymes; Event; Exons; Gene Expression; Gene-Modified; Genes; Genetic; Genetic screening method; Genomics; Goals; Growth; Guanosine Triphosphate; Guide RNA; Haloferax volcanii; Homologous Gene; Human; In Vitro; Introns; Iodine; Label; Laboratories; Lead; Ligase; Maps; Mediating; Metabolism; Methylation; Modification; Mutate; Nuclease P1; Positioning Attribute; Post-Transcriptional RNA Processing; Process; Production; Proteins; Pseudouridine; RNA; RNA Processing; RNA Splicing; RNA, Transfer, Met; Reaction; Recombinants; Regulator Genes; Reporter Genes; Research; Ribonucleases; Role; Small Nucleolar Ribonucleoproteins; System; T7 RNA polymerase; Testing; Thin Layer Chromatography; Time; Transcript; Transfer RNA; Translations; Work; Yeasts; base; crosslink; endonuclease; in vivo; inorganic phosphate; phosphorothioate; prevent; public health relevance; research study; tRNA Precursor

DESCRIPTION (provided by applicant): Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Broad long-term objectives of this application are to characterize various RNA processing events in Archaea. The specific aims of this proposal are: Structural and functional characterization of archaeal box C/D sRNPs; In vivo characterization of the production of 2'-O-methylcytidine (Cm) and 2'-O-methyluridine (Um) at positions 34 and 39, respectively, of Haloferax volcanii tRNATrp and study, in vivo, the relationship among these modification reactions, splicing and processing of pre-tRNATrp; Determination of the effect of Cm modification at the wobble position of tRNA on the accuracy of translation in H. volcanii; and Characterization of pseudouridine formation at positions 54 and 55 in archaeal tRNAs. Structural and functional studies of dual guide-target RNAs will be done by using recombinant box C/D RNP core proteins along with 32P-labeled T7 RNA polymerase generated pre-tRNA targets and their introns. Structural studies of the dynamic interaction in a sequential modification system will be studied using lead (II) induced cleavage mapping and phosphorothioate-iodine footprinting. Functional characterization of these sRNP complexes will be done using thin layer chromatography of RNase T2 or nuclease P1 digests of their in vitro reaction products. In vivo cross-linking approaches will be utilized to isolate sRNP associated proteins. In vivo characterization of sRNP guide modifications and their relationship with splicing will be studied with a modified version of tRNATrp gene. The tRNA product of this modified gene and normal genomic gene can be distinguished. Products of various mutated versions of this modified gene will be characterized in in vivo studies. Role of the intron in wobble base modification of tRNA and its effect on the accuracy of translation will be tested by using a specifically modified reporter gene and a tRNATrp intron-deleted H. volcanii strain. Finally tRNA pseudouridine synthases, Pus10 and aCbf5 will be studied in vitro and in vivo for their ability to produce pseudouridine at positions 54 and/or 55 of tRNAs. Overall the basic understanding of these processes in archaeal systems will help us understand similar processes in human systems under normal conditions and the changes that may occur under diseased conditions. PUBLIC HEALTH RELEVANCE: Studies of gene regulatory phenomena such as RNA processing help us in understanding cellular metabolism and growth under normal as well as diseased conditions. Studying these processes in complex system is often challenging and sometimes not even feasible. Using simple model organisms, we elucidate the mechanisms and functions of these events, which can then be correlated to human systems.

描述（申请人提供）：RNA转录后加工可以调节基因表达，对细胞代谢、生长和分化的控制至关重要。该应用的长期目标是表征古细菌中各种RNA加工事件。本课题的具体目标是：古细菌盒状C/D sRNPs的结构和功能表征；在体内研究了火山卤铁tRNATrp在34位和39位分别产生2′- o -甲基胞苷（Cm）和2′- o -甲基尿苷（Um），并研究了这些修饰反应、剪接和加工前tRNATrp之间的关系；测定tRNA摆动位置Cm修饰对火山螺翻译精度的影响古细菌trna中54和55位伪尿嘧啶形成的表征。利用重组盒C/D RNP核心蛋白和32p标记的T7 RNA聚合酶生成的pre-tRNA靶标及其内含子，进行双导向靶标RNA的结构和功能研究。序列修饰体系中动态相互作用的结构研究将使用铅（II）诱导解理作图和硫代磷-碘足迹进行研究。这些sRNP复合物的功能表征将使用RNase T2或核酸酶P1体外反应产物的薄层色谱进行。体内交联方法将用于分离sRNP相关蛋白。sRNP引导修饰的体内表征及其与剪接的关系将通过tRNATrp基因的修饰版本进行研究。该修饰基因的tRNA产物与正常基因组基因可以区分。该修饰基因的各种突变版本的产物将在体内研究中进行表征。内含子在tRNA的摆动碱基修饰中的作用及其对翻译准确性的影响将通过特异性修饰的报告基因和tRNATrp内含子缺失的H. volcanii菌株进行测试。最后，我们将在体外和体内研究tRNA伪尿嘧啶合成酶Pus10和aCbf5在tRNA的54和/或55位产生伪尿嘧啶的能力。总的来说，对古菌系统中这些过程的基本理解将有助于我们理解正常条件下人类系统中的类似过程以及患病条件下可能发生的变化。公共卫生相关性：基因调控现象（如RNA加工）的研究有助于我们了解正常和患病条件下的细胞代谢和生长。在复杂系统中研究这些过程往往具有挑战性，有时甚至是不可行的。使用简单的模式生物，我们阐明了这些事件的机制和功能，然后可以将其与人类系统相关联。