Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhang, Ziheng, Sun, Weiping, Shi, Tiezhu, Lu, Pengfei, Zhuang, Min, Liu, Ji-Long
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
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
_version_ 1783534836539981824
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
work_keys_str_mv AT zhangziheng capturingrnaproteininteractionviacruis
AT sunweiping capturingrnaproteininteractionviacruis
AT shitiezhu capturingrnaproteininteractionviacruis
AT lupengfei capturingrnaproteininteractionviacruis
AT zhuangmin capturingrnaproteininteractionviacruis
AT liujilong capturingrnaproteininteractionviacruis