Assessing messenger RNA decapping in cellular extracts.
Removal of the 5' cap from a messenger RNA (mRNA) is an integral part of all mRNA decay pathways and can be a highly regulated event. Assays designed to assess decapping in vitro need to effectively resolve four products of mRNA decay: 7meGpppG produced by 3'-5' shortening of the transcript by the exosome, 7meGMP produced by the scavenger decapping enzyme DcpS acting on the product of exosomal decay, 7meGDP produced by the Dcp1/2 decapping enzyme, and free phosphate, which can be generated by phosphatases in the extract acting upon either of the two products of decapping noted above. We have outlined both thin-layer chromatography and acrylamide-gel based approaches that can be used to assess decapping activities.
Bergman
Naomi
N
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA.
Milone
Joseph
J
Bates
Elizabeth J
EJ
Opyrchal
Mateusz
M
Bellofatto
Vivian
V
Wilusz
Jeffrey
J
D002855:Chromatography, Thin Layer / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004722:Endoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012315:RNA Caps; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012270:Ribosomes / Q000502:physiology
D012315:RNA Caps; D012333:RNA, Messenger; C030052:mRNA decapping enzymes; D004722:Endoribonucleases