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

Summary

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.

Abstract

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.

Introduction

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 diseases^3,4,5.

The pa....

Protocol

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 cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL st....

Results

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 .......

Discussion

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.......

Materials

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