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A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

BACKGROUND: Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fract...

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Autores principales: Zhang, Qiqi, Zhao, Fengli, Wu, Zhe, Zhu, Danling
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9743689/
https://www.ncbi.nlm.nih.gov/pubmed/36510301
http://dx.doi.org/10.1186/s13007-022-00967-y
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author Zhang, Qiqi
Zhao, Fengli
Wu, Zhe
Zhu, Danling
author_facet Zhang, Qiqi
Zhao, Fengli
Wu, Zhe
Zhu, Danling
author_sort Zhang, Qiqi
collection PubMed
description BACKGROUND: Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants. RESULT: Here, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin. CONCLUSION: This optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-022-00967-y.
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spelling pubmed-97436892022-12-13 A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis Zhang, Qiqi Zhao, Fengli Wu, Zhe Zhu, Danling Plant Methods Methodology BACKGROUND: Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants. RESULT: Here, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin. CONCLUSION: This optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-022-00967-y. BioMed Central 2022-12-12 /pmc/articles/PMC9743689/ /pubmed/36510301 http://dx.doi.org/10.1186/s13007-022-00967-y Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Methodology
Zhang, Qiqi
Zhao, Fengli
Wu, Zhe
Zhu, Danling
A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title_full A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title_fullStr A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title_full_unstemmed A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title_short A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis
title_sort simple and robust method for isolating and analyzing chromatin-bound rnas in arabidopsis
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9743689/
https://www.ncbi.nlm.nih.gov/pubmed/36510301
http://dx.doi.org/10.1186/s13007-022-00967-y
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