RNA Splicing in Archaea

转录后RNA加工可以调节基因表达，这对于基因的表达是必不可少的。 控制细胞代谢、生长和分化。研究这些复杂的过程 系统往往具有挑战性，有时甚至不可行。类真核生物通常具有 过程，但在基本层面上，从而作为更简单的模型系统，以获得深入了解 这些复杂的细胞活动。本申请的广泛长期目标是表征 各种RNA加工事件。 本提案的具体目标是：（1）确定C/D箱指南中发生的变化 在sRNP的组装期间以及在修饰反应期间和之后;（2）体内 古细菌Cbf 5蛋白和向导H/ACA RNA的结构和功能的表征;以及 (3)鉴定Pus 10蛋白质的tRNA Y 54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32 P末端标记的T7 RNA聚合酶产生的转录物的切割作图将 在重组盒C/D RNP核心蛋白存在下进行，用于指导和 靶RNA及其在催化过程中的相互作用。研究其结构和功能意义 Cbf 5蛋白和H/ACA RNA的同源性降低，相应的基因在H.火山所得 缺失菌株将用表达突变体Cbf 5的基因的质粒携带拷贝转化 蛋白质/HACARNA，并将使用CMCT和U- 具体反应。将在体外研究Pus 10的几种突变形式，以研究它们产生 在tRNA的位置54和/或55处的假尿苷。这些突变体的影响将进一步研究 使用E. coli的异源体内系统。 盒C/D和H/ACA sRNP组分中的缺陷已经涉及几个方面。 疾病因此，全面了解古细菌系统中的这些过程将有助于我们 了解正常情况下人体系统中的类似过程，以及可能发生的变化， 发生在患病的情况下。

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.