Unexpected electrophoretic migration of RNA with different 3' termini causes a RNA sizing ambiguity that can be resolved using nuclease P1-generated sequencing ladders.
It has been widely believed that the electrophoretic migration difference of otherwise identical RNAs with a P versus OH terminus would be the same as occurs for DNA, a fairly reproducible approximately 1/2 nucleotide (nt) offset. RNA with a 5'-OH indeed migrates </=1 nt slower than if it had a 5'-P. Surprisingly, however, RNA with a 3'-OH terminus (generated by many cellular RNases of interest) migrates anywhere from approximately 1/4 to approximately 2 nts slower than the otherwise identical molecule with a 3'-P or 2', 3'-cyclic-P terminus (as present on standard RNase-generated sequencing ladders). This previously unrecognized variability in electrophoretic migration offset causes a 1-2 nt ambiguity in a commonly used method of RNA size determination. We also show two ways to overcome this problem and enable rigorous sizing of 3'-OH terminating RNAs. Most convenient is to use sequencing standards generated by nuclease P1, which is generally sequence-nonspecific but we show becomes G-specific or A-preferential under certain reaction conditions.
Cruz-Reyes
J
J
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Piller
K J
KJ
Rusché
L N
LN
Mukherjee
M
M
Sollner-Webb
B
B
D000818:Animals; D001345:Autoradiography; D003573:Cytochrome b Group / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007202:Indicators and Reagents; D012313:RNA / Q000737:chemistry; D012322:RNA Precursors / Q000737:chemistry; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000737:chemistry*; D006163:Ribonuclease T1; D017422:Sequence Analysis, DNA / Q000379:methods; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014346:Trypanosoma brucei brucei / Q000235:genetics
D003573:Cytochrome b Group; D007202:Indicators and Reagents; D012322:RNA Precursors; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D012313:RNA; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases