Proteomic profiling of Arid3a 3′UTR-associated proteins using MS2-MCP-TurboID proximity labeling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Dan Lu
PROVIDER: PXD080101 | iProX | Tue Jun 23 00:00:00 BST 2026
REPOSITORIES: iProX
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Project description:This project presents a comprehensive profiling of pre-miRNA156a-interacting proteins using an integrated MCP-MS2 and TurboID proximity labeling approach in vitro. We performed fusion expression of pre-miRNA with MS2, and co-expressed MCP-TurboID. Due to the specific binding between MCP and MS2, TurboID is brought into close proximity with pre-miRNA. Upon exogenous addition of biotin, interacting molecules of pre-miRNA are biotinylated by TurboID. After cell lysis and incubation with streptavidin magnetic beads, the biotinylated proteins are enriched. Finally, mass spectrometry (MS) analysis is performed to identify the molecules interacting with pre-miRNA, thereby capturing the interaction profile of pre-miRNA-associated proteins.
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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.
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