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: ^7me GpppG produced by 3′–5′ shortening of the transcript by the exosome, ^7me GMP produced by the scavenger decapping enzyme DcpS acting on the product of exosomal decay, ^7me GDP 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 thinlayer chromatography and acrylamide-gel based approaches that can be used to assess decapping activities.