Nov 4, 2019

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

BioRxiv : the Preprint Server for Biology
Jeetayu BiswasRobert Singer

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 nascen...Continue Reading

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Mentioned in this Paper

In Vivo
ADAR
Genes
Regulation of Biological Process
Transcription, Genetic
ADAM8
Monocyte Chemoattractant Protein-1
Evaluation
Binding Protein
Health Care Systems

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