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Identification of pre-miRNA156a binding proteins by MCP-MS2/TurboID

ABSTRACT: This study developed an integrated MCP-MS2/TurboID proximity labeling system to identify pre-miRNA156a-interacting proteins in vitro. By co-expressing MS2-tagged pre-miRNA156a with MCP-TurboID fusion protein, we achieved specific recruitment of TurboID to the target RNA. Following a 10-minute biotin pulse, proximal proteins were biotinylated, enriched using streptavidin magnetic beads, and separated by SDS-PAGE. Comparative analysis of differential protein bands between experimental and control groups, followed by LC-MS/MS analysis of excised gel bands (35-55 kDa), enabled systematic identification of pre-miRNA156a-binding proteins. This approach combines the specificity of MCP-MS2 interaction with the sensitivity of TurboID-mediated proximity labeling for comprehensive RNA-protein interactome profiling.

ORGANISM(S): Arabidopsis Thaliana

SUBMITTER: Yunfeng Liu

PROVIDER: PXD066141 | iProX | Tue Jul 15 00:00:00 GMT+01:00 2025

REPOSITORIES: iProX

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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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Identification of pre-miRNA156a binding proteins by MCP-MS2/TurboID

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