Chapter 5 PABLO Analysis of RNA

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.