RNA Splicing in Archaea

古细菌中的 RNA 剪接

• 批准号: 8289877
• 负责人: Ramesh Gupta
• 金额: $ 29.1万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8289877
• 关键词: Academic Research Enhancement Awards; Address; Animal Model; Animals; Apoptosis; Archaea; Area; Award; Bacteria; Biological Models; Boxing; Catalysis; Complex; Core Protein; Defect; Differentiation and Growth; Disease; Dyskeratosis Congenita; Enzymes; Escherichia coli; Eukaryota; Event; Gene Expression; Genes; Goals; Grant; Growth; Homologous Gene; Human; Human Activities; In Vitro; Incubated; Introns; Label; Laboratories; Lead; Maps; Metabolism; Methylation; Mitochondria; Modification; Physiological; Plasmids; Polymerase; Positioning Attribute; Process; Proteins; Pseudouridine; RNA Splicing; RNA, Transfer, Met; Reaction; Recombinants; Regulator Genes; Research; Ribonucleoproteins; Ribosomal RNA; S-Adenosylmethionine; Signal Transduction; Site; Small Nucleolar RNA; Small Nucleolar Ribonucleoproteins; Structure; Structure-Activity Relationship; System; TNFSF10 gene; Techniques; Time; Transcript; Transfer RNA; Work; Yeasts; base; cytochrome c; in vivo; insight; mutant; preference; rRNA Precursor; research study

Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Studying these processes in complex systems is often challenging and sometimes not even feasible. Archaea often have eukaryote-like processes, but at a basic level, thus serving as much simpler model systems to gain insights into these complex cellular events. Broad long-term objectives of this application are to characterize various RNA processing events in Archaea. Specific aims of this proposal are: (1) Determination of the changes occurring in box C/D guide and target RNAs during assembly of sRNPs and during and after modification reactions; (2) In vivo characterization of the structure and function of archaeal Cbf5 protein and a guide H/ACA RNA; and (3) Identification of the determinants for tRNA Y54 synthase activity of Pus10 proteins. A variety of in vitro and in vivo techniques will be used to address the above-metioned aims. Lead(II) induced cleavage mapping of 32P-end-labeled T7 RNA polymerase generated transcripts will be done in presence of recombinant box C/D RNP core proteins for the structural studies of guide and target RNAs and their interactions during catalysis. To study the structural and functional significance of Cbf5 protein and H/ACA RNA, corresponding genes will be deleted in H. volcanii. The resulting deletion strains will be transformed with plasmid-borne copies of genes expressing mutant Cbf5 protein/HACA RNA, and the status of rRNA pseudouridylation will be checked using CMCT and U- specific reactions. Several mutant versions of Pus10 will be studied in vitro for their ability to produce pseudouridine at positions 54 and/or 55 of tRNAs. The effects of these mutants will be further studied using an E. coli based heterologous in vivo system. Defects in both box C/D and H/ACA sRNP components have been implicated in several diseases. Thus, the overall 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.

转录后 RNA 加工可以调节基因表达，这对于 细胞代谢、生长和分化的控制。研究这些复杂的过程 系统通常具有挑战性，有时甚至不可行。古细菌通常具有真核生物样特征 过程，但在基础层面上，因此可以作为更简单的模型系统来深入了解 这些复杂的细胞事件。该应用的广泛长期目标是表征 古细菌中的各种 RNA 加工事件。 该提案的具体目标是： (1) 确定 C/D 框指南中发生的变更 sRNP 组装期间以及修饰反应期间和之后的目标 RNA； (2) 体内 古菌 Cbf5 蛋白和指导 H/ACA RNA 的结构和功能表征；和 (3)Pus10蛋白tRNA Y54合酶活性决定因素的鉴定。 将使用各种体外和体内技术来实现上述目标。 Lead(II) 诱导的 32P 末端标记的 T7 RNA 聚合酶生成的转录物的切割图谱将 在存在重组盒 C/D RNP 核心蛋白的情况下完成，用于指导和 目标 RNA 及其在催化过程中的相互作用。研究结构和功能意义 Cbf5蛋白和H/ACA RNA的缺失，H. volcanii中相应的基因将会被删除。由此产生的 缺失菌株将用表达突变体 Cbf5 的质粒携带的基因拷贝进行转化 蛋白质/HACA RNA，并且rRNA假尿苷化的状态将使用CMCT和U-检查 具体反应。 Pus10 的几个突变版本将在体外研究其产生 tRNA 的第 54 和/或 55 位为假尿苷。这些突变体的影响将得到进一步研究 使用基于大肠杆菌的异源体内系统。 C/D 盒和 H/ACA sRNP 组件中的缺陷与多个问题有关 疾病。因此，对古细菌系统中这些过程的整体理解将帮助我们 了解正常条件下人类系统中的类似过程，以及可能发生的变化 发生在患病条件下。