MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing

Abstract

Summary Both UV-cross-linking and immunoprecipitation (CLIP) and RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives of CLIP and TRIBE, endogenous β-actin mRNA was tagged with MS2 stem loops, making it the only bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE detected β-actin, albeit with false-positives. False-positive CLIP signals were attributed to nonspecific antibody interactions. In contrast, putative false-positive TRIBE targets were genes spatially proximal to the β-actin gene. MCP-ADAR edited nearby nascent transcripts consistent with interchromosomal contacts observed in Hi-C. The identification of nascent contacts implies RNA regulatory proteins (e.g., splicing factors) associated with multiple nascent transcripts, forming domains of post-transcriptional activity. Repeating these results with an integrated inducible MS2 reporter indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts to study spatial organization and nuclear RNA regulation.

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“…Computational analysis was performed as outlined in Rahman et al [ 62 ] to identify editing events present in Fne-ADAR CD and ADAR CD . The thresholds used in Biswas et al [ 63 ] were applied here, namely a minimum of 20 reads and a minimum editing frequency of 5%. As expected, the ADAR CD control yielded very few editing events (100–300 edits), whereas Fne-ADAR CD produced >1200 editing events in each of two replicates.…”

Protocol for using TRIBE to study RNA-protein interactions and nuclear organization in mammalian cells

“…( Biswas et al., 2020 ; Lionnet et al., 2011 ) N/A Chemicals, peptides, and recombinant proteins EDTA Sigma Cat# EDS-100G Ethanol 70% vol/vol made from 100% vol/vol ethanol and ultrapure water Thermo Fisher Scientific Cat# P288-500 jetPRIME transfection reagent Polyplus Cat# 114-15 TRIzol LS Reagent Thermo Fisher Scientific Cat# 10296010 TURBO DNA-free Kit Thermo Fisher Scientific AM1907 Critical commercial assays NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with Sample Purification Beads New England Biolabs Cat# E7765 NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) New England Biolabs Cat# E7335 PCR Purification Kit Thermo Fisher Scientific Cat# K310001 Qubit RNA HS Assay Kit Thermo Fisher Scientific Cat#Q32852 Plasmid DNA Midiprep/Maxiprep Kit MACHEREY-NAGEL Cat#740410 or Cat#740414 Deposited data MS2-TRIBE data, raw and analyzed data This study. ( Biswas et al., 2020 ) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152855 Experimental models: cell lines Human osteosarcoma, U2OS ( Janicki et al., 2004 ) N/A Immortalized mouse embryonic fbroblasts, MEFs This study. ( Biswas et al., 2020 ; Lionnet et al., 2011...…”

“…( Biswas et al., 2020 ) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152855 Experimental models: cell lines Human osteosarcoma, U2OS ( Janicki et al., 2004 ) N/A Immortalized mouse embryonic fbroblasts, MEFs This study. ( Biswas et al., 2020 ; Lionnet et al., 2011 ) N/A Recombinant DNA Plasmid: mCherry-ADAR Addgene https://www.addgene.org/154786/ Plasmid: MCP-ADAR Addgene https://www.addgene.org/154787/ Software and algorithms BEDTools ( Quinlan and Hall, 2010 ) https://github.com/arq5x/bedtools2 Bowtie2 ( Langmead and Salzberg, 2012 ) http://bowtie-bio.sourceforge.net/bowtie2/index.shtml Cutadapt ( Martin, 2011 ) https://cutadapt.readthedocs.io/en/stable/installation.html FastQC Babraham Bioinformatics https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ GraphPad Prism, version 8 GraphPad https://www.graphpad.com/scientific-software/prism/ HTSeq, version 0.6.1 ( Anders et al., 2015 ) https://github.com/htseq/htseq HyperTRIBE software ( Biswas et al...…”

“…Necessary steps for performing a TRIBE experiment are detailed, starting with plasmid/cell line generation, cellular transfection, and RNA sequencing library preparation and concluding with bioinformatics analysis of RNA editing sites and identification of target RNAs. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Biswas et al. (2020) .…”