RNA Splicing in Archaea

古细菌中的 RNA 剪接

• 批准号: 6954447
• 负责人: Ramesh Gupta
• 金额: $ 21.68万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954447
• 关键词: Archaea; DNA directed RNA polymerase; Halobacteriaceae; RNA splicing; biochemical evolution; cytidine; gel mobility shift assay; introns; methylation; microorganism genetics; oligonucleotides; protein binding; recombinant proteins; reporter genes; ribonucleoproteins; small nuclear ribonucleoproteins; spliceosomes; thin layer chromatography; transfer RNA; tryptophan; uridine

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 AREA application are to characterize various RNA processing events in Archaea. Both Archaea and Bacteria are prokaryotes; yet Archaea exhibit several molecular features resembling Eukaryotes. This proposal specifically deals with processing of intron-containing pre-tRNAs in Haloferax volcanii, a halophilic archaeon. The specific aims of this proposal are: Characterization of in vitro sRNP system for site-specific modifications in H. volcanii pre-tRNATrp; Characterization, in vivo, of the production of 2'-O-methylcytidine (Cm) and 2'-O-methyluridine (Um) at positions 34 and 39, respectively, of the tRNATrp in H. volcanii and study, in vivo, the relationship between these modification reactions and pre-tRNA splicing; and Determination of the effect of Cm modification at the wobble position of tRNA on the accuracy of translation in H. volcanii. Mechanism of 2'-O-methylation of H. volcanii pre-tRNATrp nucleotides, in vitro, will be studied by using recombinant box C/D RNP core proteins and H. volcanii cell extracts along with 32P-labeled T7 RNA polymerase generated pre-tRNAs and their introns. Binding of core proteins to normal and variously modified substrates will be determined by gel-shift assays. Production of the 2'-O-methylated nucleotides will be determined by thin layer chromatography (TLC) of the RNase T2 or nuclease P1 digests of the RNAs. A modified version of tRNATrp gene will be used to study, in vivo, box C/D guided modifications reactions and their relationship to splicing. The tRNA product of this modified gene and normal genomic gene can be distinguished. Products of variously mutated versions of this modified gene will be characterized in in vivo studies. Extracts of the cells containing mutated forms of this modified gene will also be used for the in vitro methylation studies. Role of the intron in wobble base modification of tRNA and its effect on the accuracy of translation will be tested by using specifically modified reporter gene and an intron-deleted tRNA gene. The tRNA products of this intronless gene will be characterized for the presence or absence of specific Cm and Um modifications by separating their RNase T1 digests by denaturing gels or fingerprinting, and determining the composition of the appropriate oligonucleotides by TLC.

描述（由申请人提供）：转录后RNA加工可以调节基因表达，这对于控制细胞代谢、生长和分化至关重要。该 AREA 应用的广泛长期目标是表征古生菌中的各种 RNA 加工事件。古细菌和细菌都是原核生物；然而古细菌表现出一些类似于真核生物的分子特征。该提案具体涉及嗜盐古菌 Haloferax volcanii 中含有内含子的前 tRNA 的加工。 该提案的具体目标是： 表征 H. volcanii pre-tRNATrp 中位点特异性修饰的体外 sRNP 系统；体内表征H. volcanii中tRNATrp分别在位置34和39处产生2'-O-甲基胞苷（Cm）和2'-O-甲基尿苷（Um），并在体内研究这些修饰反应与前tRNA剪接之间的关系；确定 tRNA 摆动位置处的 Cm 修饰对 H. volcanii 翻译准确性的影响。 将使用重组盒 C/D RNP 核心蛋白和火山嗜血菌细胞提取物以及 32P 标记的 T7 RNA 聚合酶生成前 tRNA 及其内含子，在体外研究火山嗜血杆菌前 tRNATrp 核苷酸的 2'-O-甲基化机制。核心蛋白与正常和不同修饰的底物的结合将通过凝胶迁移测定来确定。 2'-O-甲基化核苷酸的产生将通过 RNA 的 RNase T2 或核酸酶 P1 消化物的薄层色谱 (TLC) 来确定。 tRNATrp 基因的修饰版本将用于体内研究盒 C/D 引导的修饰反应及其与剪接的关系。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。含有该修饰基因突变形式的细胞提取物也将用于体外甲基化研究。内含子在 tRNA 碱基摆动修饰中的作用及其对翻译准确性的影响将通过使用专门修饰的报告基因和内含子缺失的 tRNA 基因进行测试。该无内含子基因的 tRNA 产物将通过变性凝胶或指纹分析分离其 RNase T1 消化物，并通过 TLC 确定适当寡核苷酸的组成，来表征是否存在特定的 Cm 和 Um 修饰。