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PubMed · 14770006

Assessing messenger RNA decapping in cellular extracts.

Naomi Bergman·Joseph Milone·Elizabeth J Bates·Mateusz Opyrchal·Vivian Bellofatto·Jeffrey Wilusz
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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.

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Chromatography, Thin LayerElectrophoresis, Polyacrylamide GelEndoribonucleasesHeLa CellsHumansPeptide Chain Initiation, TranslationalProtein BiosynthesisRNA CapsRNA, MessengerRibosomesJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Naomi Bergman, Joseph Milone, Elizabeth J Bates, Mateusz Opyrchal, Vivian Bellofatto, Jeffrey Wilusz. 2004. Assessing messenger RNA decapping in cellular extracts.. https://doi.org/10.1385/1-59259-750-5%3A181

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