Studies of RNA modifications on the genomic scale

描述（由申请人提供）： 已经发现了96种不同类型的转录后核糖核苷酸修饰。它们存在于所有三个生命界的tRNA、rRNA中，以及真核生物中的mRNA、小核RNA和其他RNA中。43 E。仅大肠杆菌tRNA就含有25种类型的300多个修饰位点。tRNA修饰的结构和功能已经研究了很长时间。在这300个站点中，只有不到25%的站点具有已知的功能。据我们所知，基本上所有关于转录后RNA修饰的功能研究都集中在单个位点或单个类型的修饰上。这项建议将开发微阵列方法，同时监测所有E。coli tRNAs。微阵列系统将用于监测各种环境条件下所有tRNA修饰的定量分数的变化及其对体内tRNA氨酰化效率的影响。第一个目标是设计、打印和测试tRNA微阵列，以检测和量化所有tRNA修饰的程度。我们将合成新的寡核苷酸探针，对个别类型的修饰敏感。结合我们以前开发的tRNA微阵列系统，这些新的寡核苷酸探针将被打印在微阵列上，并测试它们在全基因组范围内检测tRNA修饰和定量测定所有位点的修饰分数的有效性。第二个目标是检测大肠杆菌中tRNA的修饰和氨酰化的效率。杆菌我们目前的tRNA微阵列系统同时测量所有tRNA的氨酰化分数。目前的方法将适用于测量氨酰化分数作为体内位点特异性tRNA修饰程度的函数。这些测量将揭示所有修饰在基因组水平上的协调及其对tRNA基本功能的组合效应。

DESCRIPTION (provided by applicant): 96 different types of post-transcriptional ribonucleotide modifications have been discovered. They are present in tRNA, rRNA in all three kingdoms of life, and in mRNA, small nuclear RNA and other RNAs in eukaryotes. The 43 E. coli tRNAs alone contain more than 300 sites of modifications of 25 types. The structure and function of tRNA modifications have been studied for a very long time. Less than 25% of these 300 sites have known functions. To our knowledge, essentially all functional studies on post-transcriptional RNA modifications focused on, a single site or a single type of modifications. This proposal will develop microarray methods that simultaneously monitor known modifications in all E. coli tRNAs. The microarray system will be used to monitor changes in the quantitative fraction of all tRNA modifications under a variety of environmental conditions and their effects on the efficiency of tRNA aminoacylation in vivo. The first aim will design, print, and test tRNA microarrays that detect and quantify the extent of all tRNA modifications. We will synthesize new oligonucleotide probes that are sensitive to individual types of modifications. Combined with our previously developed tRNA microarray system, these new oligonucleotide probes will be printed on microarrays and tested for their effectiveness in genome-wide detection of tRNA modifications and the quantitative determination on the fraction of modifications at all sites. The second aim will measure tRNA modifications and the efficiency of aminoacylation in E. coli. Our current tRNA microarray system simultaneously measures the fraction of aminoacylation for all tRNAs. This current method will be adapted to measure the aminoacylation fraction as a function of the extent of site-specific tRNA modifications in vivo. These measurements shall reveal the coordination of all modifications at the genomic level and their combinatorial effects on an essential function of tRNA.