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

View ORCID ProfileJeetayu Biswas, Reazur Rahman, Varun Gupta, Michael Rosbash, View ORCID ProfileRobert H. Singer
doi: https://doi.org/10.1101/829606
Jeetayu Biswas
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA
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Reazur Rahman
2Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, MA, 02453, USA
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Varun Gupta
3Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA
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Michael Rosbash
2Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, MA, 02453, USA
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Robert H. Singer
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA
4Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, 20147, USA
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  • For correspondence: robert.singer@einstein.yu.edu
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Abstract

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.

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Posted November 04, 2019.
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MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing
Jeetayu Biswas, Reazur Rahman, Varun Gupta, Michael Rosbash, Robert H. Singer
bioRxiv 829606; doi: https://doi.org/10.1101/829606
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MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing
Jeetayu Biswas, Reazur Rahman, Varun Gupta, Michael Rosbash, Robert H. Singer
bioRxiv 829606; doi: https://doi.org/10.1101/829606

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