Detection and quantitation of RNA base modifications

Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5' side of a nucleotide of interest. Next, (32)p is substituted for the phosphate at the 5' end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by Phosphorlmager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. WE! also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U-34 is similar to90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.