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Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, th...

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Autores principales: Forero-Quintero, Linda S., Raymond, William, Handa, Tetsuya, Saxton, Matthew N., Morisaki, Tatsuya, Kimura, Hiroshi, Bertrand, Edouard, Munsky, Brian, Stasevich, Timothy J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155019/
https://www.ncbi.nlm.nih.gov/pubmed/34039974
http://dx.doi.org/10.1038/s41467-021-23417-0
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author Forero-Quintero, Linda S.
Raymond, William
Handa, Tetsuya
Saxton, Matthew N.
Morisaki, Tatsuya
Kimura, Hiroshi
Bertrand, Edouard
Munsky, Brian
Stasevich, Timothy J.
author_facet Forero-Quintero, Linda S.
Raymond, William
Handa, Tetsuya
Saxton, Matthew N.
Morisaki, Tatsuya
Kimura, Hiroshi
Bertrand, Edouard
Munsky, Brian
Stasevich, Timothy J.
author_sort Forero-Quintero, Linda S.
collection PubMed
description The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.
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spelling pubmed-81550192021-06-11 Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene Forero-Quintero, Linda S. Raymond, William Handa, Tetsuya Saxton, Matthew N. Morisaki, Tatsuya Kimura, Hiroshi Bertrand, Edouard Munsky, Brian Stasevich, Timothy J. Nat Commun Article The carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5. Nature Publishing Group UK 2021-05-26 /pmc/articles/PMC8155019/ /pubmed/34039974 http://dx.doi.org/10.1038/s41467-021-23417-0 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Forero-Quintero, Linda S.
Raymond, William
Handa, Tetsuya
Saxton, Matthew N.
Morisaki, Tatsuya
Kimura, Hiroshi
Bertrand, Edouard
Munsky, Brian
Stasevich, Timothy J.
Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title_full Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title_fullStr Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title_full_unstemmed Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title_short Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene
title_sort live-cell imaging reveals the spatiotemporal organization of endogenous rna polymerase ii phosphorylation at a single gene
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155019/
https://www.ncbi.nlm.nih.gov/pubmed/34039974
http://dx.doi.org/10.1038/s41467-021-23417-0
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