Advances in Ide.pngication of RNA-binding Proteins Based on Mass Spectrometry

doi:10.13523/j.cb.2211057

China Biotechnology

2023, Vol. 43

Issue (7): 77-87 DOI: 10.13523/j.cb.2211057

Advances in Ide.pngication of RNA-binding Proteins Based on Mass Spectrometry

Ruo-hang SUN,Rui-bing CHEN^**(

)

School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China

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Abstract

RNA-binding proteins (RBPs) can bind RNAs through RNA-binding domains and regulate the fate or functions of their bound RNAs. RBPs are responsible for all the steps of RNA metabolism throughout their life cycle and are involved in many other cellular processes related to cell’s survival, replication, and adaption to environmental changes. RNA-protein interactions are essential to cell homeostasis, and their defects are associated with various diseases. Therefore, the characterization of RBPs is crucial to depict their functions and underlying molecular mechanisms. Recently, several methods have been developed and extensively applied to ide.pngy RBPs, uncovering many previously unannotated RBPs. However, the majority of the newly discovered RBPs lack classical RNA-binding domains, thus highlighting the complexity and diversity of RNA-protein interactions. Here, the progress in the ide.pngication of RBPs based on mass spectrometry was reviewed, from two aspects: high-throughput ide.pngication and targeted ide.pngication. Moreover, their technical principles, application areas, and strengths and limitations were discussed, providing new perspectives to better understand the biological significance and clinical implications of RNA-protein interactions.

Key words： RNA-binding protein RNA-protein interaction Proteomics Mass spectrometry

Received: 29 November 2021 Published: 03 August 2023

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Ruo-hang SUN, Rui-bing CHEN. Advances in Ide.pngication of RNA-binding Proteins Based on Mass Spectrometry. China Biotechnology, 2023, 43(7): 77-87.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2211057 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I7/77

Fig.1 Schematic diagram of RIC strategy

Fig.2 Schematic diagram of high-throughput ide.pngication of the RBPs based on RNA labeling (a) RBR-ID ide.pngies RBPs by quantitative proteomics based on the decrease in the mass spectrometric intensities of the RBPs after UV-CL (b) 5-EU metabolically labeled RNA are crosslinked to RBPs, and the RNA-protein complexes containing 5-EU are linked with azide-biotin via click chemistry reaction to enable affinity purification with SA-coated beads (e.g., RICK and CARIC) (c) RNAs are labeled by PP under UV irradiation, and then the crosslinked RNA-protein complexes are purified by SA-coated beads

Fig.3 Schematic diagram of high-throughput ide.pngication of the RBPs based on physicochemical properties (a) Biphasic extraction strategy uses AGPC to isolate crosslinked RNA-protein complexes based on their solubility (e.g., XRNAX and OOPS) (b) TRAPP isolates UV-crosslinked RNA-protein complexes based on their silica affinity (c) Ide.pngication of RBPs based on their RNase-sensitive MW shift (e.g., R-DeeP and DIF-FRAC)

Fig.4 Schematic diagram of the targeted ide.pngication of RBPs in vitro (a) RNA tags are used to purify the target RNA and its binding proteins by affinity beads. After incubation with cell lysates, target RNA-binding proteins are eluted and analyzed by mass spectrometry (b) Fluorescence labeled RNAs are hybridized to a protein microarray to detect and qua.pngy RNA binding via fluorescence measurement

Fig.5 Schematic diagram of the targeted ide.pngication of RBPs in vivo (a) Biotinylated antisense nucleotide probes are used to isolate the target RNA and associated proteins after UV or chemical crosslinking (e.g., CHART, ChIRP, and RAP) (b) Aptamers incorporated target RNAs are expressed in the cells to isolate the binding proteins (e.g., MS2-BioTRAP, RaPID, and TOBAP-MS) (c) dCas13 fused to a proximity-labeling enzyme are recruited to target RNA by gRNA, and RBPs are biotinylated and isolated with SA-coated beads (e.g., CRUIS and CARPID)

Table 1 List of the high-throughput and targeted methods for ide.pngication of RBPs


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