Proximity labeling to detect RNA-protein interactions in live cells.

Mingxing Lu,Wencheng Wei

doi:10.1002/2211-5463.12706

Mingxing Lu, Wencheng Wei

Open Access

https://doi.org/10.1002/2211-5463.12706

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Abstract

RNA biology is orchestrated by the dynamic interactions of RNAs and RNA‐binding proteins (RBPs). In the present study, we describe a new method of proximity‐dependent protein labeling to detect RNA–protein interactions [RNA‐bound protein proximity labeling (RBPL)]. We selected the well‐studied RNA‐binding protein PUF to examine the current proximity labeling enzymes birA* and APEX2. A new version of birA*, BASU, was used to validate that the PUF protein binds its RNA motif. We further optimized the RBPL labeling system using an inducible expression system. The RBPL (λN‐BASU) labeling experiments exhibited high signal‐to‐noise ratios. We subsequently determined that RBPL (λN‐BASU) is more suitable than RBPL (λN‐APEX2) for the detection of RNA–protein interactions in live cells. Interestingly, our results also reveal that proximity labeling is probably capable of biotinylating proximate nascent peptide.

Highlights

RNA biology is orchestrated by the dynamic interactions of RNAs and RNA-binding proteins (RBPs)
We describe a new method of proximity-dependent protein labeling to detect RNA–protein interactions [RNA-bound protein proximity labeling (RBPL)]
BoxB RNA motif recruits the RBPL protein, thereby biotinylating proteins bound to the flanked adjacent RNA motif of interest (Fig. 1A), allowing capture by streptavidin of RNA motif-bound proteins for analysis by western blotting or MS

Summary

Introduction

RNA biology is orchestrated by the dynamic interactions of RNAs and RNA-binding proteins (RBPs). This method only identifies highly abundant RNAs and isolates interactions that are cross-linked in cells It requires a huge quantity of starting materials to obtain high purification yields of RNA–protein complexes because any individual RNA is probably present at only a very small proportion of the total cellular RNAs. Chromatin immunoprecipitation has been used to determine chromatin bound DNA motifs. Abbreviations APEX, ascorbate peroxidase; BASU, birA*, from Bacillus subtilis; bioID, proximity-dependent biotin identification; CLIP, cross-linking immunoprecipitation; RAP, RNA antisense purification; RBPL, RNA-bound protein proximity labeling; RBPs, RNA-binding proteins; RIP, RNA immunoprecipitation. RBPL detects RNA–protein interactions in live cells immunoprecipitation-derived methods have been proposed for the detection of RBPs, termed RNA immunoprecipitation (RIP) [16,17,18,19,20,21]. In CLIP-seq, UV cross-linking is more specific, it only links proteins to RNAs that are at near-zero distance [23]

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