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.
Celesnik
Helena
H
Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA
Deana
Atilio
A
Belasco
Joel G
JG
D001483:Base Sequence; D015152:Blotting, Northern; D011756:Diphosphates / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011756:Diphosphates; D012333:RNA, Messenger