Determination of posttranscriptional modification in RNA by mass spectrometry

Substantial biochemical and genetic evidence exists that implicates ribosomal RNA as a functional component of the translational apparatus. The nucleotide sequences in functionally important regions of ribosomal RNA have defined features of universal conservation, single-stranded ness [as defined by secondary structure], and surface proximity [as defined by tertiary or quaternary structure]. Specific nucleotides within these regions are posttranscriptionally modified, yet little in known about the chemical identity of these modifications, and almost nothing is known about their function. This paucity of information stems from the lack of practical methodologies required to elucidate chemical structure of posttranscriptionally modified nucleotides as a prerequisite to investigation of structure/function relationships. A method for the detection, chemical characterization, and sequence location of posttranscriptionally modified nucleotides in RNA is presented. The method is based on the ability to infer the base composition of an oligonucleotide, simple by accurate measurement of molecular mass by electrospray mass spectrometry. Posttranscriptional modifications are recognized from incremental increases in mass, In conjunction with combined liquid chromatography/thermospray mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level and assigned to sequence sites within oligonucleotides defined by selective ribonuclease cleavage. The method is demonstrated using Escherichia coli 5S rRNA, in which all oligonucleotides produced by ribonuclease T1 hydrolysis are identified solely on the basis of their molecular masses, and using Escherichia coli 16S rRNA, in which several posttranscriptionally modified oligonucleotides were characterized. The method is used to characterized, 5,6-dihydrouridine at position 2449 in the highly conserved central loop of domain V in Escherichia coli 23S ribosomal RNA, a region which has been strongly implicated in the peptidyl transferase activity.