CBRPP: a new RNA-centric method to study RNA-protein interactions

Abstract

0RNA-protein interactions play essential roles in tuning gene expression at RNA level and 1 1 modulating the function of proteins. Abnormal RNA-protein interactions lead to cell dysfunction 1 2 and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for 1 3 understanding cellular mechanism and pathogenesis of diseases. Different practical protein-centric 1 4 methods for studying RNA-protein interactions has been reported, but few RNA-centric methods 1 5 exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new 1 6RNA-centric method to identify proteins associated with the target RNA in native cellular context 1 7 without cross-linking or RNA manipulation in vitro. CBRPP is based on a fusion of dCas13 and 1 8 proximity-based labeling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with 1 9 high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are 2 0 then identified by mass spectrometry. 2 1 Keywords: RNA-protein interactions, dPspCas13b, dRfxCas13d, APEX2, TurboID, BASU, 2 2 BioID2, CBRPP 2 3 Introduction 2 4RNA is bound to protein from birth to death. RNA-binding proteins (RBPs) play a pivotal role 2 5 in a wide range of biological processes, including RNA transcription, processing, modification, 2 6 transport, translation and stabilization [1][2][3][4] . RNAs, in turn, influence proteins expression, 2 7 localization and interactions with other proteins [5][6][7] . Aberrant RNA-protein interactions are related 2 8to cellular dysfunction and human diseases [3, 8, 9] . Therefore, mapping networks of RNA-protein 2 9interactions is of great importance for understanding many cellular biological processes. 0Based on the type of molecule they start with, methods for studying RNA-protein interactions 3 1 are classified into protein-centric methods and RNA-centric methods [10] . Protein-centric methods 3 2 start with a protein of interest and study RNAs that interact with that protein. Since proteins are 3 3 easily purified with antibodies, many protein-centric methods, such as cross-linking 3 4 immunoprecipitation (CLIP)-seq [11] and RNA immunoprecipitation (RIP)-seq [12] , are available and 3 5 practical. Conversely, RNA-centric methods start with an RNA of interest and focus on proteins 3 6 that bind it. Most approaches use biotinylated RNA [13] , aptamer-tagged RNA [14] , peptide nucleic 3 7 acid [15] and antisense probe [16][17][18][19][20] for purification of RNA-protein complexes to identify proteins 3 8 that associate with the target RNA, however these methods often require RNA manipulation in 3 9 vitro and miss transient or weak interactions. Meanwhile, compared with protein-centric methods, 4 0 there are few robust RNA-centric methods. 4 1 In this paper, by combining the power of CRISPR-Cas13 [21] and proximity-based labeling (PBL) 4 2 technique [22] , we developed CBRPP (CRISPR-based RNA proximity proteomics), a new 4 3 RNA-centric method to identify proteins associated with the target RNA in native c...

In vivo discovery of RNA proximal proteins 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 labelling enzyme (APEX2, BioID2, BASU, and PafA) were reported [112][113][114][115]. 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.…”

In vivodiscovery 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.…”

CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research

“…In this regard, several groups have fused dCas13 proteins with proximity labeling enzymes to investigate RNA-protein interactions in living cells (Table ). When the dCas13 was targeted to any RNA, the labeling enzyme catalyzed the ligation of biotin − (or a biotin-labeled protein) to RNA-binding proteins, which are then captured with streptavidin (Figure H). Therefore, dCas13-based methods offer powerful tools for RNA-protein interaction mapping and can also be used to study RNA-protein interactions in cancer cells.…”