RNA Splicing in Archaea

描述(申请人提供)：转录后RNA处理可以调节基因表达，这对于控制细胞新陈代谢、生长和分化是必不可少的。这项申请的广泛的长期目标是表征古生代的各种RNA加工事件。该建议的具体目的是：鉴定古菌盒C/D sRNPs的结构和功能；在体内鉴定Haloferax cancanii tRNATrp在第34和39位分别产生2‘-O-甲基胞苷(Cm)和2’-O-甲基尿苷(Um)的产物，并在体内研究这些修饰反应、剪接和前tRNATrp加工之间的关系；确定tRNA摆动位置的Cm修饰对H.Volcanii翻译准确性的影响；以及鉴定古生物tRNAs中54和55位的伪尿苷形成。双引导靶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聚合酶产生的前tRNA靶标及其内含子来完成。将使用铅(II)诱导的裂解图谱和硫代磷-碘足迹来研究序列修饰系统中动态相互作用的结构研究。这些rRNP复合体的功能特征将使用RNaseT2或核酸酶P1的薄层层析进行，这些RNaseT2或核酸酶P1消化其体外反应产物。体内交联法将被用来分离sRNP相关蛋白。将用tRNATrp基因的修改版本来研究sRNP引导修饰的体内特征及其与剪接的关系。该修饰基因的tRNA产物与正常基因组基因的tRNA产物可以区分。这种修饰基因的各种突变版本的产物将在体内研究中确定其特征。内含子在tRNA摆动碱基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改的报告基因和tRNATrp内含子缺失的火山杆菌菌株来测试。最后，将在体外和体内研究tRNA假性尿苷合成酶Pus10和aCbf5在tRNAs 54和/或55位产生假性尿苷的能力。总体而言，对古生物系统中这些过程的基本了解将有助于我们理解人类系统在正常条件下的类似过程以及在疾病条件下可能发生的变化。公共卫生相关性：对RNA加工等基因调控现象的研究有助于我们理解正常和疾病条件下的细胞新陈代谢和生长。在复杂系统中研究这些过程往往是具有挑战性的，有时甚至是不可行的。利用简单的模型生物，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联。

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.