Capturing RNA-protein interaction via CRUIS

Nucleic Acids Research
Ziheng ZhangJi-Long Liu

Abstract

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.

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Citations

Sep 11, 2020·Wiley Interdisciplinary Reviews. Developmental Biology·Justin A BoschNorbert Perrimon
Aug 26, 2020·Proceedings of the National Academy of Sciences of the United States of America·Shuo HanAlice Y Ting
Jan 26, 2021·Biochemical Society Transactions·Jeffrey M SmithAndrew I Webb
Jan 13, 2021·Nature Methods·Wei QinAlice Y Ting
Apr 4, 2021·International Journal of Molecular Sciences·Vesper Burjoski, Anireddy S N Reddy
May 1, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ronja WeissingerOrit Hermesh
Apr 27, 2021·Plant Communications·Xinxin YangYongliang Zhang
Jun 22, 2021·Bio-protocol·Weiping SunMin Zhuang
Jul 14, 2021·Advanced Science·Vanja PerčulijaSongying Ouyang
Apr 30, 2020·Biochemistry·Mingxing Lu, Taku A Tokuyasu
Sep 24, 2021·Wiley Interdisciplinary Reviews. RNA·Martyna KordyśZbigniew Warkocki
Sep 30, 2021·Molecular Microbiology·Jessica KimmelTobias Spielmann
Oct 19, 2021·Plant Physiology·Andrea Mair, Dominique C Bergmann

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Methods Mentioned

BETA
immunoprecipitation
RIP
pulldown
transfection
affinity-based purification
ChIP

Software Mentioned

NORAD
R package Limma
CRUIS
R package clusterProfiler
CRIUS
Addgene
ggplot2
Metascape
GLoPro
R

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