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Elizabeth J Bates

Publications and source records attributed to Elizabeth J Bates.

2 recordsLinked to original sources
PubMed2004

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: 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.

Chromatography, Thin Layer↗
PubMed2002

Analysis of the products of mRNA decapping and 3'-to-5' decay by denaturing gel electrophoresis.

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.

Chromatography, Thin Layer↗