Capturing RNA–protein interaction via CRUIS

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.

In vivo discovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

“…During the preparation of our manuscript, similar strategies using different fusion proteins of endonuclease-deficient Cas13 protein (dLwaCas13a, dPspCas13b, and dRfxCas13d) and proximity labeling enzyme (APEX2, BioID2, BASU, and PafA) were reported (Han et al, 2020; Li et al, 2020; Yi et al, 2020; Zhang et al, 2020). Applications of these methods together with ours to both mRNAs and ncRNAs with wide range of abundance (∼10 2 -10 6 copies/cell) demonstrate that these methods have broad potential to identify functional relevant RBPs for diverse transcripts.…”

RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

“…The recently developed CRISPR-based RNA targeting systems provide a new option to directly label RBPs in living cells without the need for crosslinking procedures [ 89 , 90 ]. For instance, RNA-United Interacting System (CRUIS) captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT to frame a proximity targeting system [ 89 ].…”

“…The recently developed CRISPR-based RNA targeting systems provide a new option to directly label RBPs in living cells without the need for crosslinking procedures [ 89 , 90 ]. For instance, RNA-United Interacting System (CRUIS) captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT to frame a proximity targeting system [ 89 ]. Specifically, the dead RNA-guided RNA targeting nuclease LwaCas13a (dLwaCas13a) is used as a tracker to target specific RNA sequences, while the proximity labeling enzyme PafA fused to dLwaCas13a labels any surrounding RBP.…”

EVLncRNAs 2.0: an updated database of manually curated functional long non-coding RNAs validated by low-throughput experiments

“…The ‘binding’ indicates that lncRNA has direct physical contact with other biomolecules. State-of-the-art methods such as ChIRP ( 26 ), CHART ( 27 ), RAP ( 28 ), HyPR ( 29 ), CRUIS ( 30 ), CLIP ( 31 , 32 ) or CLASH ( 33 ) make screening of the direct physical binding interactions between lncRNAs and proteins, RNAs or DNAs. However, whether or not these interactions have any biological significance require further low-throughput experimental validations.…”

“…Now users can search for a specific lncRNA, disease or lncRNA-interacting molecule within a specific drop-down option (lncRNAs, Diseases, Interactions) in addition to the previously provided keyword-based search. The keyword search now includes the first round of screening experimental methods such as ChIRP ( 26 ), CHART ( 27 ), RAP ( 28 ), HyPR ( 29 ), CRUIS ( 30 ), CLIP ( 31 , 32 ) and CLASH ( 33 ) in addition to low-throughput techniques. The ‘Tools’ page now includes EVlncRNA-pred for predicting the likelihood of a given lncRNA from high-throughput expression experiments to be functional ( 41 ), Context Fold ( 42 ) and SPOT-RNA ( 43 ) for RNA secondary structure prediction, and RNAsnap2 for RNA solvent accessibility prediction ( 44 ).…”