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Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells

Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we...

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Autores principales: Douka, Katerina, Agapiou, Michaela, Birds, Isabel, Aspden, Julie L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8822167/
https://www.ncbi.nlm.nih.gov/pubmed/35145996
http://dx.doi.org/10.3389/fmolb.2021.791455
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author Douka, Katerina
Agapiou, Michaela
Birds, Isabel
Aspden, Julie L.
author_facet Douka, Katerina
Agapiou, Michaela
Birds, Isabel
Aspden, Julie L.
author_sort Douka, Katerina
collection PubMed
description Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we tested a variety of conditions, which contribute to the preciseness of ribosome footprinting and therefore the success of ribosome profiling. We found that NaCl concentration, RNaseI source, RNaseI amount, and temperature of footprinting all contributed to the quality of ribosome footprinting in human neuroblastoma SH-SY5Y cells. These ideal conditions for footprinting also improved footprint quality when used with Drosophila melanogaster S2 cells. Footprinting under the same conditions generated different footprints sizes and framing patterns in human and D. melanogaster cells. We also found that treatment of S2 cells with cycloheximide prior to footprinting impacted the distribution of footprints across ORFs, without affecting overall read length distribution and framing pattern, as previously found in other organisms. Together our results indicate that a variety of factors affect ribosome footprint quality and the nature of precise footprinting varies across species.
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spelling pubmed-88221672022-02-09 Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells Douka, Katerina Agapiou, Michaela Birds, Isabel Aspden, Julie L. Front Mol Biosci Molecular Biosciences Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we tested a variety of conditions, which contribute to the preciseness of ribosome footprinting and therefore the success of ribosome profiling. We found that NaCl concentration, RNaseI source, RNaseI amount, and temperature of footprinting all contributed to the quality of ribosome footprinting in human neuroblastoma SH-SY5Y cells. These ideal conditions for footprinting also improved footprint quality when used with Drosophila melanogaster S2 cells. Footprinting under the same conditions generated different footprints sizes and framing patterns in human and D. melanogaster cells. We also found that treatment of S2 cells with cycloheximide prior to footprinting impacted the distribution of footprints across ORFs, without affecting overall read length distribution and framing pattern, as previously found in other organisms. Together our results indicate that a variety of factors affect ribosome footprint quality and the nature of precise footprinting varies across species. Frontiers Media S.A. 2022-01-25 /pmc/articles/PMC8822167/ /pubmed/35145996 http://dx.doi.org/10.3389/fmolb.2021.791455 Text en Copyright © 2022 Douka, Agapiou, Birds and Aspden. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Douka, Katerina
Agapiou, Michaela
Birds, Isabel
Aspden, Julie L.
Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title_full Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title_fullStr Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title_full_unstemmed Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title_short Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells
title_sort optimization of ribosome footprinting conditions for ribo-seq in human and drosophila melanogaster tissue culture cells
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8822167/
https://www.ncbi.nlm.nih.gov/pubmed/35145996
http://dx.doi.org/10.3389/fmolb.2021.791455
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