The extensive post-transcriptional modification of tRNA is required for proper translation of the genetic code into protein, and defects in tRNA modifications can cause disease. To detect tRNA modifications that could impact human health we are utilizing primer extension as a sensitive approach to detect these modifications in cell culture. Primer extension readily detects modifications such as N2,N2 dimethylguanosine (m2,2G), because this modification sits on the Watson-Crick face of the nucleotide, resulting in a primer extension block. Other modifications, such as 2’-O-methylation, do not interfere with base-pairing, and therefore require additional treatment of the RNA prior to primer extension analysis. In one approach, treatment of RNA with the SHAPE reagent N-methyl-isatoic anahydride (NMIA) generates a large adduct on the 2’-hydroxl group of the residue which interferes with Watson-Crick pairing. However, nucleotides with a 2’-O-methlyation are not modified by NMIA, allowing for detection of the modification as loss of a primer extension block. As a proof of principle that primer extension can be used to identify a tRNA modification in cell culture, the putative gene required for m2,2G modification in Drosophila melanogaster was knocked down in D. melanogaster S2R+ cells using double-stranded RNA (dsRNA) treatment. As expected, this dsRNA treatment of cells resulted in a specific loss of a primer extension block on target tRNAs. Once a reliable assay to detect 2’-O-methylation is developed, it will be used in combination with RNA knockdown and bioinformatics to identify new tRNA methylation genes in cultured animal and human cells.

Development of a sensitive assay to detect tRNA modifications in cell culture

253rd National Meeting (2017)

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