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

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 toba...

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Autores principales: Paquette, David R., Mugridge, Jeffrey S., Weinberg, David E., Gross, John D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769751/
https://www.ncbi.nlm.nih.gov/pubmed/29101277
http://dx.doi.org/10.1261/rna.062737.117
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author Paquette, David R.
Mugridge, Jeffrey S.
Weinberg, David E.
Gross, John D.
author_facet Paquette, David R.
Mugridge, Jeffrey S.
Weinberg, David E.
Gross, John D.
author_sort Paquette, David R.
collection PubMed
description 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.
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spelling pubmed-57697512019-02-01 Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping Paquette, David R. Mugridge, Jeffrey S. Weinberg, David E. Gross, John D. RNA Method 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. Cold Spring Harbor Laboratory Press 2018-02 /pmc/articles/PMC5769751/ /pubmed/29101277 http://dx.doi.org/10.1261/rna.062737.117 Text en © 2018 Paquette et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Paquette, David R.
Mugridge, Jeffrey S.
Weinberg, David E.
Gross, John D.
Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title_full Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title_fullStr Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title_full_unstemmed Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title_short Application of a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme for transcription start site mapping
title_sort application of a schizosaccharomyces pombe edc1-fused dcp1–dcp2 decapping enzyme for transcription start site mapping
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769751/
https://www.ncbi.nlm.nih.gov/pubmed/29101277
http://dx.doi.org/10.1261/rna.062737.117
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