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处理可以调节基因表达，这对于 控制细胞代谢，生长和分化。研究这些过程中的复杂过程 系统通常具有挑战性，有时甚至不可行。古细菌经常像真核生物一样 流程，但在基本层面，因此提供了更简单的模型系统，以了解 这些复杂的细胞事件。该应用程序的长期目标是表征 古细菌的各种RNA处理事件。 该提案的具体目的是：（1）确定方框C/D指南中发生的更改 在SRNP和修饰反应期间和之后的组装过程中靶向RNA； （2）体内 古细菌CBF5蛋白的结构和功能的表征和指导H/ACA RNA；和 （3）鉴定PUS10蛋白的tRNA Y54合酶活性的决定因素。 各种体外和体内技术将用于解决上述目标。 铅（ii）诱导的32p端标记的T7 RNA聚合酶产生的转录本的切割图将 在存在重组盒C/D RNP核心蛋白的存在下完成指导的结构研究和 靶向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的相应基因将在H. tolcanii中删除。结果 缺失菌株将用表达突变体CBF5的基因的质粒传播拷贝转化 蛋白质/HACA RNA以及RRNA伪苷的状态将使用CMCT和U-检查 具体反应。将在体外研究几种突变版本的PUS10，以生产其能力 pseudouridine位于trnas的位置54和/或55。这些突变体的影响将进一步研究 使用基于大肠杆菌的异源体内系统。 两个方框C/D和H/ACA SRNP组件中的缺陷均与几个 疾病。因此，对古细菌系统中这些过程的总体理解将帮助我们 了解正常条件下人类系统中类似的过程，以及可能的变化 在患病的情况下发生。