β-Actin mRNA interactome mapping by proximity biotinylation

Mukherjee, Joyita; Hermesh, Orit; Eliscovich, Carolina; Nalpas, Nicolas C.; Franz‐Wachtel, Mirita; Maček, Boris; Jansen, Ralf‐Peter

doi:10.1073/pnas.1820737116

Cited by 66 publications

(47 citation statements)

References 66 publications

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“…Recently, proteins that interact with the β -actin-MBS were profiled using MCP fused to the biotin ligase BirA. Interestingly, several nuclear proteins and chromatin components were discovered even after control subtraction, suggesting a similar co-transcriptional contribution to RNA-protein profiling when the MBS array is used (Mukherjee et al, 2019). However, TRIBE differs in the mechanism of transcriptional marking: the enzyme is affixed to the transcript rather than an interaction that occurs by chance diffusion of a reactive intermediate.…”

Section: Identification Of Chromatin Contacts and Transcription Domainsmentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

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Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking ("CLIP" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP ("TRIBE"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β -actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β -actin gene. This result indicates that MCP-ADAR bound to β -actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.3 Introduction:

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Section: Identification Of Chromatin Contacts and Transcription Domainsmentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

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“…The key advantage of PBL is that it can capture weak and transient interactions in live cells. Recently, two studies have applied PBL to study RNA-protein interactions using the MS2-MCP strategy [28] or a similar strategy [27] , but both require insertion of MS2 or BoxB stem-loop into the target RNA in advance, which may influence structure or function of the target RNA.…”

Section: Resultsmentioning

confidence: 99%

“…Compared with previous RNA-centric methods, CBRPP has several advantages. First, CBRPP does not require pre-labeling of the target RNA [13] , MS2 insertion in advance [28] , or designing antisense probes [16–20] to purify RNA-protein complexes. In dPspCas13b-BioID2 positive cells only crRNAs are required.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Cao

et al. 2020

Preprint

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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...

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“…An important limitation of our approach and other existing proximity labeling RNA-protein interaction mapping methods 12,13 is that they have only been demonstrated on overexpressed, highly-abundant cellular RNAs. Based on previous studies using MCP-GFP for single-molecule imaging of MS2-tagged mRNAs [79][80][81] , it is likely that our MCP-APEX2 strategy should have sufficient sensitivity to be extensible to lowerabundance RNAs, albeit with an increase in number of MS2 step loops (to >20 stem loops, compared to the 4 used here).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, unbiased RNA detection is generally easier than unbiased detection of proteins due to the wide availability of RNA sequencing methods. To address the challenge of RNA-centered interactome mapping, recent studies 12,13 have started to employ proximitydependent labeling 14 , targeting the promiscuous biotin ligase BioID 15 to specific MS2-or BoxB stem-looptagged RNAs. However, the low catalytic efficiency of BioID necessitates very long labeling times (18-24 hours), precluding the study of dynamic processes, while the stem-loop tags could affect the function of target RNAs.…”

Section: Introductionmentioning

confidence: 99%

RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

Han

Zhao

Myers

et al. 2020

Preprint

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RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. Capitalizing on the ability of the engineered peroxidase APEX2 to identify protein interaction partners via proximity-dependent biotinylation, we used two approaches to target APEX2 to specific cellular RNAs. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were able to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured endogenous protein interaction partners of hTR, including more than a dozen proteins not previously linked to hTR. We validated the unexpected interaction between hTR and the N 6 -methyladenosine (m 6 A) demethylase ALKBH5. Further investigation showed that endogenous hTR is modified by m 6 A, which can be erased by ALKBH5, and that ALKBH5 influences both telomerase complex assembly and activity. These results highlight the ability of MS2-and Cas13-targeted APEX2 to identify novel RNA-protein interactions in living cells.

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β-Actin mRNA interactome mapping by proximity biotinylation

Cited by 66 publications

References 66 publications

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

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

RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

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