Mapping pseudouridines in RNA molecules

Pseudouridine is present in ribosomal RNA, transfer RNA, tmRNA, and small nuclear and nucleolar RNAs. All are structured molecules. Pseudouridine is made by enzyme-catalyzed isomerization of specifically selected U residues after the polynucleotide chain is made. No energy input is required. Pseudouridine formation creates a hydrogen bond donor at the equivalent of uridine C-5. Therefore, a major role of pseudouridine may be to strengthen particular RNA conformations and/or RNA-RNA interactions because of this extra H-bond capability. Understanding the role of pseudouridine critically depends on knowledge of their location and number in RNA. The mapping method described here has greatly simplified this task and made it possible to survey many organisms. Procedures are described for mapping pseudouridines in large RNAs like ribosomal RNA and in small RNAs like tRNA. The method involves carbodiimide adduct formation with U, G, and pseudouridine followed by mild alkali to remove the adduct from U and G but not from the N-3 of pseudouridine. This results in attenuation of primed reverse transcription resulting in a stop band one residue 3' to the pseudouridine on sequencing gels. Use of primers means that purified RNAs are not needed, only knowledge of its primary sequence, but limit the sequence scanned to 30-40 residues from the 3' end. A poly(A) tailing procedure is described that allows extension of the method to within a few nucleotides of the 3' terminus.