TY - JOUR
T1 - Assessing messenger RNA decapping in cellular extracts.
AU - Bergman, Naomi
AU - Milone, Joseph
AU - Bates, Elizabeth J.
AU - Opyrchal, Mateusz
AU - Bellofatto, Vivian
AU - Wilusz, Jeffrey
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=2142827899&partnerID=8YFLogxK
U2 - 10.1385/1-59259-750-5:181
DO - 10.1385/1-59259-750-5:181
M3 - Article
C2 - 14770006
AN - SCOPUS:2142827899
SN - 1064-3745
VL - 257
SP - 181
EP - 192
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -