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The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells
7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m(6)A). Currently available 5′-ca...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038993/ https://www.ncbi.nlm.nih.gov/pubmed/31984425 http://dx.doi.org/10.1093/nar/gkaa032 |
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author | Sikorski, Pawel J Warminski, Marcin Kubacka, Dorota Ratajczak, Tomasz Nowis, Dominika Kowalska, Joanna Jemielity, Jacek |
author_facet | Sikorski, Pawel J Warminski, Marcin Kubacka, Dorota Ratajczak, Tomasz Nowis, Dominika Kowalska, Joanna Jemielity, Jacek |
author_sort | Sikorski, Pawel J |
collection | PubMed |
description | 7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m(6)A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m(7)GpppN((m))pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m(6)A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/A(m) and m(6)A(m), whereas the lowest was observed for G and G(m). The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells. |
format | Online Article Text |
id | pubmed-7038993 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-70389932020-03-02 The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells Sikorski, Pawel J Warminski, Marcin Kubacka, Dorota Ratajczak, Tomasz Nowis, Dominika Kowalska, Joanna Jemielity, Jacek Nucleic Acids Res NAR Breakthrough Article 7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m(6)A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m(7)GpppN((m))pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m(6)A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/A(m) and m(6)A(m), whereas the lowest was observed for G and G(m). The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells. Oxford University Press 2020-02-28 2020-01-27 /pmc/articles/PMC7038993/ /pubmed/31984425 http://dx.doi.org/10.1093/nar/gkaa032 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | NAR Breakthrough Article Sikorski, Pawel J Warminski, Marcin Kubacka, Dorota Ratajczak, Tomasz Nowis, Dominika Kowalska, Joanna Jemielity, Jacek The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title | The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title_full | The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title_fullStr | The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title_full_unstemmed | The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title_short | The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells |
title_sort | identity and methylation status of the first transcribed nucleotide in eukaryotic mrna 5′ cap modulates protein expression in living cells |
topic | NAR Breakthrough Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038993/ https://www.ncbi.nlm.nih.gov/pubmed/31984425 http://dx.doi.org/10.1093/nar/gkaa032 |
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