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Live imaging of transcription sites using an elongating RNA polymerase II–specific probe
In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 ph...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8647360/ https://www.ncbi.nlm.nih.gov/pubmed/34854870 http://dx.doi.org/10.1083/jcb.202104134 |
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author | Uchino, Satoshi Ito, Yuma Sato, Yuko Handa, Tetsuya Ohkawa, Yasuyuki Tokunaga, Makio Kimura, Hiroshi |
author_facet | Uchino, Satoshi Ito, Yuma Sato, Yuko Handa, Tetsuya Ohkawa, Yasuyuki Tokunaga, Makio Kimura, Hiroshi |
author_sort | Uchino, Satoshi |
collection | PubMed |
description | In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains. |
format | Online Article Text |
id | pubmed-8647360 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-86473602021-12-20 Live imaging of transcription sites using an elongating RNA polymerase II–specific probe Uchino, Satoshi Ito, Yuma Sato, Yuko Handa, Tetsuya Ohkawa, Yasuyuki Tokunaga, Makio Kimura, Hiroshi J Cell Biol Tools In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains. Rockefeller University Press 2021-12-02 /pmc/articles/PMC8647360/ /pubmed/34854870 http://dx.doi.org/10.1083/jcb.202104134 Text en © 2021 Uchino et al. https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Tools Uchino, Satoshi Ito, Yuma Sato, Yuko Handa, Tetsuya Ohkawa, Yasuyuki Tokunaga, Makio Kimura, Hiroshi Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title | Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title_full | Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title_fullStr | Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title_full_unstemmed | Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title_short | Live imaging of transcription sites using an elongating RNA polymerase II–specific probe |
title_sort | live imaging of transcription sites using an elongating rna polymerase ii–specific probe |
topic | Tools |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8647360/ https://www.ncbi.nlm.nih.gov/pubmed/34854870 http://dx.doi.org/10.1083/jcb.202104134 |
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