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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

Published: October 19th, 2018

DOI:

10.3791/58580

1College of Chemistry and Molecular Engineering, Peking University, 2Beijing National Laboratory for Molecular Sciences, Peking University, 3Peking-Tsinghua Center for Life Sciences, Peking University, 4Synthetic and Functional Biomolecules Center, Peking University, 5Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University
* These authors contributed equally

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

The human genome is transcribed into various types of coding and noncoding RNAs (ncRNAs), including mRNAs, rRNAs, tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and long non-coding RNAs (lncRNAs)1. Most of these RNAs possess clothing of RBPs and function as ribonucleoprotein particles (RNPs)2. Therefore, a comprehensive identification of RBPs is a prerequisite for understanding the regulatory network between RNAs and RBPs, which is implicated in various human diseases3,4,5.

The pa....

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CAUTION: When applicable, the reagents used should be purchased in the form of RNase-free, or dissolved in RNase-free, solvents (for most cases, in diethyl pyrocarbonate (DEPC)-treated water). When handling RNA samples and RNase-free reagents, always wear gloves and masks, and change them frequently to avoid RNase contamination.

1. Preparation of Lysate of Metabolically Labeled and UV Cross-linked Cells

  1. Metabolic incorporation of EU and 4SU
    1. Culture HeLa.......

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The representative results of quality control steps are presented. The results include figures of the in-gel fluorescence analysis described in step 2.3.2 (Figure 1), the western blot analysis described in step 4.1.3 (Figure 2A), and the silver-staining analysis described in step 4.2.2 (Figure 2B). The quality control steps are critical for the optimization of CARIC protocols. Always include quality .......

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The maintenance of fair RNA integrity is one of the keys to successful CARIC experiments. With appropriate ligands of Cu(I) and careful operation, RNA degradation can be significantly reduced, although partial degradation was observed. The substitution ratios of EU and 4SU in experimental samples are 1.18% and 0.46%, respectively (data not shown). For intact RNAs with a length of 2,000 nt, ~90% of RNAs contain at least one EU and one 4SU. For partially degraded RNAs with a length of 1,000 nt, ~70% of RNAs contain at leas.......

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This work is supported by the National Natural Science Foundation of China Grants 91753206, 21425204, and 21521003 and by the National Key Research and Development Project 2016YFA0501500.

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Name Company Catalog Number Comments
HeLa ATCC
DMEM (Dulbecco's Modified Eagle Medium) Thermo Fisher Scientific 11995065
FBS (Fetal Bovine Serum) Thermo Fisher Scientific 10099141
Penicillin & Streptomycin Thermo Fisher Scientific 15140122
EU (5-ethynyl uridine) Wuhu Huaren Co. CAS:69075-42-9
4SU (4-thiouridine) Sigma Aldrich T4509
10×PBS (Phosphate-Buffered Saline) Thermo Fisher Scientific AM9625
UV cross-linker UVP CL-1000 Equiped with 365-nm UV lamp
DEPC (Diethyl pyrocarbonate) Sigma Aldrich D5758 To treat water. Highly toxic!
Tris·HCl, pH 7.5 Thermo Fisher Scientific 15567027
LiCl Sigma Aldrich 62476
Nonidet P-40 Biodee 74385
EDTA-free protease inhibitor cocktail Thermo Fisher Scientific 88265 One tablet for 50 mL lysis buffer.
LDS (Lithium dodecyl sulfate) Sigma Aldrich L9781
15-mL ultrafiltration tube (10 kDa cutoff) Millipore UFC901024
0.5-mL ultrafiltration tube (10 kDa cutoff) Millipore UFC501096
Streptavidin magnetic beads Thermo Fisher Scientific 88816
DMSO (Dimethyl sulfoxide) Sigma Aldrich 41639
Azide-biotin Click Chemistry Tools AZ104
Copper(II) sulfate Sigma Aldrich C1297
THPTA [Tris(3-hydroxypropyltriazolylmethyl)amine] Sigma Aldrich 762342
Sodium ascorbate Sigma Aldrich 11140
Azide-Cy5 Click Chemistry Tools AZ118
LDS sample buffer (4×) Thermo Fisher Scientific NP0008
10% bis-Tris gel Thermo Fisher Scientific NP0301BOX
EDTA Thermo Fisher Scientific AM9260G
RNase A Sigma Aldrich R6513
SDS (Sodium dodecyl sulfate) Thermo Fisher Scientific 15525017
NaCl Sigma Aldrich S3014
Brij-97 [Polyoxyethylene (20) oleyl ether] J&K 315442
Triethanolamine Sigma Aldrich V900257
Streptavidin agarose Thermo Fisher Scientific 20353
Urea Sigma Aldrich U5378
Sarkosyl (N-Lauroylsarcosine sodium salt) Sigma Aldrich 61743
Biotin Sigma Aldrich B4501
Sodium deoxycholate Sigma Aldrich 30970
MaxQuant Version: 1.5.5.1

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