title: Assessing messenger RNA decapping in cellular extracts.
abstract: 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.
authors.affiliation: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Bergman
authors.forename: Joseph
authors.initials: J
authors.lastname: Milone
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Bates
authors.forename: Mateusz
authors.initials: M
authors.lastname: Opyrchal
authors.forename: Vivian
authors.initials: V
authors.lastname: Bellofatto
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wilusz
mesh_terms: 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
chemical_list: D012315:RNA Caps; D012333:RNA, Messenger; C030052:mRNA decapping enzymes; D004722:Endoribonucleases