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Nucleic Acids Research, 1993, Vol. 21, No. 19 4577-4585
© 1993

RNA

A novel method for the determination of posttranscriptional modification in RNA by mass spectrometry

Jeffrey A. Kowalak1, Steven C. Pomerantz2, Pamela F. Crain2 and James A. McCloskey1,2,*

1Departments of Biochemistry, University of Utah Salt Lake City,UT 84112, USA 2Departments of Medicinal chemistry, University of Utah Salt Lake City,UT 84112, USA

To whom correspondence should be addresses at: Department of Medicinal Chemistry, 311A skaggs hall, University of Utah, salt Lake City, Ut&112, USA

Received June 1, 1993. Revised August 16, 1993. Accepted August 16, 1993.

A method is described for the detection, chemical characterization and sequence placement of posttranscriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-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 RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T, fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of posttranscriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.


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