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（由申请人提供）：转录后RNA处理可以调节基因表达，这对于控制细胞代谢，生长和分化至关重要。该应用程序的广泛长期目标是表征古细菌中各种RNA处理事件。该提案的具体目的是：古细胞盒C/D SRNP的结构和功能表征；在体内表征2'-O-甲基辛丁定（CM）和2'-O-甲基尿素（UM）在34和39位置分别由Haloferax Volcanii trnatrp和研究，在体内，这些修饰反应，PRICICATION REAVESS，PRICICATION和TRNNATRP PRNNATRP的处理之间的关系；确定CM修饰在tRNA的摇摆位置对H. of tRNA的准确性的影响；以及在古细胞TRNA中位置54和55处伪源形成的表征。双重指南目标RNA的结构和功能研究将通过使用重组盒C/D RNP核心蛋白以及32p标记的T7 RNA聚合酶生成的预启动靶标及其内含子来完成。将使用铅（II）诱导的裂解映射和磷酸胸酯 - 碘足迹研究顺序修饰系统中动态相互作用的结构研究。这些SRNP复合物的功能表征将使用其体外反应产物的RNase T2或核酸酶P1消化的薄层色谱法进行。体内交联方法将用于分离SRNP相关的蛋白质。 SRNP指南修饰的体内表征及其与剪接的关系将使用TRNATRP基因的修改版本进行研究。可以区分该修饰基因和正常基因组基因的tRNA产物。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Wobble碱基修饰中的作用及其对翻译准确性的影响将通过使用特定修饰的报告基因和TRNATRP内含子固定的H.火山菌菌株来测试。最后，将在体外和体内研究tRNA假氨酸合成酶，PUS10和ACBF5，以便在TRNA的位置54和/或55的位置产生假喹啉的能力。总体而言，对古细菌系统中这些过程的基本理解将有助于我们了解正常情况下人类系统中的类似过程以及在患病状况下可能发生的变化。公共卫生相关性：基因调节现象的研究，例如RNA处理，有助于我们理解正常和患病状况下的细胞代谢和生长。在复杂系统中研究这些过程通常具有挑战性，有时甚至不可行。使用简单的模型生物，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。