Dataset Information

HyperTRIBE analysis of the m6A readers in hESCs

ABSTRACT: TRIBE/HyperTRIBE has been developed as an alternative method for transcriptome-wide mapping of the RNA targets of RBPs. This method involves fusing an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR. When these fusion proteins are expressed in target cells, they direct A-to-I editing at residues in proximity to RNA binding sites. The control plasmid, mammalian expression plasmid, containing mCherry ADAR control and p2A GFP reporter can be obtained from Addgene (Plasmid #154786). As for RBP-TRIBE plasmid, using standard restriction enzyme-based cloning to clone RBP (DDX6, FUBP3, FXR2 or L1TD1) into TRIBE plasmid in hESCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE288304 | GEO | 2026/02/09

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description: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 hyperactive mutant catalytic domain of ADAR covalently attached to a specific RBP (“HyperTRIBE”). Both CLIP and TRIBE 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 in the β-actin 3’ UTR; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and HyperTRIBE (hereafter referred to as 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 interactions. However, to our surprise the putative 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. These results were repeated in human osteosarcoma cells with a randomly integrated and inducible MS2 reporter and indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts. 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 indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.

Dataset Information

HyperTRIBE analysis of the m6A readers in hESCs

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets