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RNA. 2018 Feb;24(2):251-257. doi: 10.1261/rna.062737.117. Epub 2017 Nov 3.

Application of a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme for transcription start site mapping.

Author information

1
Integrative Program in Quantitative Biology, Graduate Group in Biophysics, University of California, San Francisco, California 94158, USA.
2
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, USA.
3
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA.
4
Sandler Faculty Fellows Program, University of California, San Francisco, California 94158, USA.

Abstract

Changes in the 5' leader of an mRNA can have profound effects on its translational efficiency with little effect on abundance. Sequencing-based methods to accurately map the 5' leader by identifying the first transcribed nucleotide rely on enzymatic removal of the 5' eukaryotic cap structure by tobacco acid pyrophosphatase (TAP). However, commercial TAP production has been problematic and has now been discontinued. RppH, a bacterial enzyme that can also cleave the 5' cap, and Cap-Clip, a plant-derived enzyme, have been marketed as TAP replacements. We have engineered a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme that functions as a superior TAP replacement. It can be purified from E. coli overexpression in high yields using standard biochemical methods. This constitutively active enzyme is four orders of magnitude more catalytically efficient than RppH at 5' cap removal, compares favorably to Cap-Clip, and the 5' monophosphorylated RNA product is suitable for standard RNA cloning methods. This engineered enzyme is a better replacement for TAP treatment than the current marketed use of RppH and can be produced cost-effectively in a general laboratory setting, unlike Cap-Clip.

KEYWORDS:

Dcp2; RppH; decapping enzymes; mRNA caps; mapping mRNA 5′ ends; transcript leaders

PMID:
29101277
PMCID:
PMC5769751
DOI:
10.1261/rna.062737.117
[Indexed for MEDLINE]
Free PMC Article

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