title: Analysis of the products of mRNA decapping and 3'-to-5' decay by denaturing gel electrophoresis.
abstract: The majority of mRNA turnover is mediated either by mRNA decapping/5'-to-3' decay or exosome-mediated 3'-to-5' exonucleolytic decay. Current assays to assess mRNA decapping in vitro using cap-labeled RNA substrates rely on one-dimensional thin layer chromatography. This approach does not, however, resolve free phosphate from 7meGDP, the product of Dcp1p-mediated mRNA decapping. This can result in misinterpretation of the levels of mRNA decapping due to the generation of free phosphate following the action of the unrelated scavenger decapping activity on the products of exosome-mediated decay. In this report, we describe a simple denaturing acrylamide gel-based assay that faithfully resolves all of the possible products that can be generated from cap-labeled RNA substrates by turnover enzymes present in cell extracts. This approach allows a one-step assay to quantitatively assess the contributions of the exosome and DCP-1-type decapping on turnover of an RNA substrate in vitro. We have applied this assay to recalculate the effect of competition of cap-binding proteins on decapping in yeast. In addition, we have used the assay to confirm observations made on regulated mRNA decapping in mammalian extracts that contain much higher levels of exosome activity than yeast extracts.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, International Center for Public Health, Newark 07103, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Bergman
authors.forename: Mateusz
authors.initials: M
authors.lastname: Opyrchal
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Bates
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wilusz
mesh_terms: D002855:Chromatography, Thin Layer; D006367:HeLa Cells; D006801:Humans; D009691:Nucleic Acid Denaturation; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D012315:RNA Caps; D012331:RNA, Fungal; D012333:RNA, Messenger