Studies of RNA modifications on the genomic scale

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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.