PABLO ANALYSIS of RNA: 5′-PHOSPHORYLATION STATE AND 5′-END MAPPING

Recent studies have revealed that 5′-end-dependent RNA degradation in prokaryotes is triggered by pyrophosphate removal from the 5′-terminus to generate a monophosphorylated intermediate that is readily degraded. This chapter describes how to examine the 5′-phosphorylation state of any specific bacterial RNA by PABLO analysis. The method is based on the ability of monophosphorylated, but not triphosphorylated, RNA 5′-ends to undergo splinted ligation to a DNA oligonucleotide when juxtaposed by base pairing to a bridging oligonucleotide. PABLO analysis not only makes it possible to quantify the proportion of a particular RNA that is monophosphorylated in bacterial cells but also provides a more reliable method than primer extension for high-resolution mapping of RNA 5′-termini. © 2008 Elsevier Inc. All rights reserved.