RNA Modification in Archaea and Eukaryotes

 描述（由申请人提供）：转录后RNA加工和修饰可以调节基因表达，这对于控制细胞代谢、生长和分化至关重要。在复杂系统中研究这些过程往往具有挑战性，有时甚至不可行。古生物通常具有类似真核生物的过程，但在基本水平上。因此，它们可以作为更简单的模型系统来深入了解复杂的真核生物事件。本申请的广泛的长期目标是表征古生物中的各种RNA修饰事件，并将研究扩展到真核生物。 本发明的具体目的是：（1）在盒C/D和盒H/ACA引导RNA介导的修饰过程中，表征引导RNA和靶RNA的不同结构元件;（2）在修饰反应过程中，确定不同的盒C/D和盒H/ACA核心蛋白彼此之间以及与RNA的相互作用;（3）古生菌sRNP核心蛋白相关辅助蛋白的鉴定与鉴定;（4）真核生物特异性tRNA中Pus 54的特征和Pus 10蛋白在其合成中的作用的确定。 各种体外和体内技术将用于解决上述目标。将使用突变盒C/D和H/ACA核心蛋白进行铅（II）诱导的切割作图和32 P标记转录物的EMSA，用于RNP组装期间和催化期间向导和靶RNA的结构研究。体外修饰反应将与这些研究平行进行。不同组分的体内相互作用 通过在相应的基因缺失菌株中表达突变蛋白和RNA来研究RNP的表达。His标记的核心蛋白以及含有适体的盒C/D RNA的体内交联，随后进行H. volcanii细胞提取物将用于分离sRNP相关蛋白。分离的蛋白质将通过质谱法鉴定，并在确定其身份后进一步表征。存在或不存在在细胞RNA中的甲基化将通过CMCT和U特异性反应后的引物延伸来确定，并且2 '-O-甲基化将通过在有限的dNTP浓度下的引物延伸来确定。将测试Pus 10敲低HeLa细胞的提取物是否降低了tRNA β 54活性。将在这些细胞中表达质粒携带的Pus 10基因拷贝，以尝试挽救任何观察到的活性降低。 在疾病和感染中已经观察到RNA修饰模式和修饰机制的缺陷和变化。因此，我们对不同RNA修饰过程的研究将有助于我们了解正常条件下的这些过程，以及在疾病条件下可能发生的这些过程的任何变化。

 DESCRIPTION (provided by applicant): Post-transcriptional RNA processing and modifications 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. Therefore, they can serve as much simpler model systems to gain insights into complex eukaryotic events. Broad long-term objectives of this application are to characterize various RNA modification events in Archaea and extend the studies to eukaryotes. Specific aims of this proposal are: (1) Characterization of different structural elements of guide and target RNAs during box C/D and box H/ACA guide RNP-mediated modifications; (2) Determination of the interactions of different box C/D and box H/ACA core proteins with each other and with RNAs during modification reactions; (3) Identification and characterization of the accessory proteins associated with core proteins of archaeal guide sRNPs; and (4) Characterization of 54 in specific eukaryotic tRNAs and determination of the role of Pus10 protein in its synthesis. Various in vitro and in vivo techniques will be used to address the above-mentioned aims. Lead(II) induced cleavage mapping and EMSA of 32P-labeled transcripts will be done using mutant box C/D and H/ACA core proteins for the structural studies of guide and target RNAs during RNP assembly and during catalysis. In vitro modification reactions will be done in parallel with these studies. In vivo interactions of different components of RNPs will be studied by expressing mutant proteins and RNAs in corresponding gene-deleted strains of Haloferax volcanii. In vivo cross-linking of His-tagged core proteins as well as an aptamer-containing box C/D RNA followed by Ni-NTA chromatography of H. volcanii cell extracts will be done to isolate sRNP-associated proteins. Isolated proteins will be identified by mass spectrometry and further characterized after determining their identity. Presence or absence of ¿ in cellular RNAs will be determined by primer extension following CMCT and U-specific reactions, and 2'-O-methylation will be determined by primer extension under limited dNTP concentrations. Extracts of Pus10 knock down HeLa cells will be tested for any reduction in tRNA 54 activity. Expression of a plasmid-borne copy of the Pus10 gene in these cells will be done to attempt rescue of any observed reduced activity. Defects and changes in RNA modification patterns and modification machinery have been observed in diseases and infections. Therefore, our studies of different RNA modification processes will help us understand these processes under normal conditions, and any changes in these processes that may occur under diseased conditions.