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Capturing RNA–protein interaction via CRUIS
No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interact...
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/PMC7229851/ https://www.ncbi.nlm.nih.gov/pubmed/32140725 http://dx.doi.org/10.1093/nar/gkaa143 |
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author | Zhang, Ziheng Sun, Weiping Shi, Tiezhu Lu, Pengfei Zhuang, Min Liu, Ji-Long |
author_facet | Zhang, Ziheng Sun, Weiping Shi, Tiezhu Lu, Pengfei Zhuang, Min Liu, Ji-Long |
author_sort | Zhang, Ziheng |
collection | PubMed |
description | No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology. |
format | Online Article Text |
id | pubmed-7229851 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-72298512020-05-21 Capturing RNA–protein interaction via CRUIS Zhang, Ziheng Sun, Weiping Shi, Tiezhu Lu, Pengfei Zhuang, Min Liu, Ji-Long Nucleic Acids Res Methods Online No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology. Oxford University Press 2020-05-21 2020-03-06 /pmc/articles/PMC7229851/ /pubmed/32140725 http://dx.doi.org/10.1093/nar/gkaa143 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Zhang, Ziheng Sun, Weiping Shi, Tiezhu Lu, Pengfei Zhuang, Min Liu, Ji-Long Capturing RNA–protein interaction via CRUIS |
title | Capturing RNA–protein interaction via CRUIS |
title_full | Capturing RNA–protein interaction via CRUIS |
title_fullStr | Capturing RNA–protein interaction via CRUIS |
title_full_unstemmed | Capturing RNA–protein interaction via CRUIS |
title_short | Capturing RNA–protein interaction via CRUIS |
title_sort | capturing rna–protein interaction via cruis |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229851/ https://www.ncbi.nlm.nih.gov/pubmed/32140725 http://dx.doi.org/10.1093/nar/gkaa143 |
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