Name: Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy (Video) | JoVE Journal
Uploaded: 2018-10-19T14:00:00.000+00:00
Duration: 9 min 36 s
Description: Learn the CARIC method to identify RNA-binding proteins in HeLa cells using click chemistry and UV cross-linking. This protocol captures proteins bound to coding and noncoding RNAs for post-transcriptional gene regulation studies.

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5 minutes

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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</ol>

The authors have nothing to disclose.

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...

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 past&#160;few years have witnessed a great boost of RBPs discovered in various eukaryotic systems2,6, including human7,8,9,10,11, mouse12,13,14, yeast9,15,16, zebrafish17, Drosophila melanogaster18,19, Caenorhabditis elegans16, Arabidopsis thaliana20,21,22, and human parasites23,24,25. These advances have been facilitated by an RBP capture strategy developed by Castello et al.7 and Baltz et al.8 in 2012, which combines in vivo UV cross-linking of RNA and its interacting proteins, oligo(dT) capture of poly(A) RNAs, and mass spectrometry (MS)-based proteomic profiling. However, given the fact that poly(A) mostly exists on mature mRNAs, which account for only ~3% - 5% of the eukaryotic transcriptome26, this widely used strategy is not capable of capturing RBPs interacting with non-poly(A) RNAs, including most ncRNAs and pre-mRNAs.
Here, we report the detailed procedures of a recently developed strategy for the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs27. Termed CARIC, this strategy combines in vivo UV cross-linking and metabolic labeling of RNAs with photoactivatable and &#34;clickable&#34; nucleoside analogs (which contain a bioorthogonal functional group that can participate in click reaction), 4-thiouridine (4SU), and 5-ethynyluridine (EU). Steps that are key to get ideal results with the CARIC strategy are efficient metabolic labeling, UV cross-linking and click reaction, and the maintenance of RNA integrity. Because Cu(I) used as the catalyst in click reaction can cause the fragmentation of RNAs, a Cu(I) ligand that can reduce RNA fragmentation is essential. We describe how to perform efficient click reactions in cell lysates without causing severe RNA degradation.
Although RBP capture and identification in HeLa cells only is described in this protocol, the CARIC strategy can be applied to various cell types and possibly to living organisms. Besides RBP capture, this protocol also provides streamlined step-by-step procedures for MS sample preparation and protein identification and quantification, which can be helpful for those who are not familiar with proteomic experiments.

<table><tbody><tr><td>HeLa</td><td>ATCC</td><td></td><td></td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Thermo Fisher Scientific</td><td>10099141</td><td></td></tr><tr><td>Penicillin &amp;amp; Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>EU (5-ethynyl uridine)</td><td>Wuhu Huaren Co.</td><td>CAS:69075-42-9</td><td></td></tr><tr><td>4SU (4-thiouridine)</td><td>Sigma Aldrich</td><td>T4509</td><td></td></tr><tr><td>10&amp;times;PBS (Phosphate-Buffered Saline)</td><td>Thermo Fisher Scientific</td><td>AM9625</td><td></td></tr><tr><td>UV cross-linker</td><td>UVP</td><td>CL-1000</td><td>Equiped with 365-nm UV lamp</td></tr><tr><td>DEPC (Diethyl pyrocarbonate)</td><td>Sigma Aldrich</td><td>D5758</td><td>To treat water. Highly toxic!</td></tr><tr><td>Tris&amp;middot;HCl, pH 7.5</td><td>Thermo Fisher Scientific</td><td>15567027</td><td></td></tr><tr><td>LiCl</td><td>Sigma Aldrich</td><td>62476</td><td></td></tr><tr><td>Nonidet P-40</td><td>Biodee</td><td>74385</td><td></td></tr><tr><td>EDTA-free protease inhibitor cocktail</td><td>Thermo Fisher Scientific</td><td>88265</td><td>One tablet for 50 mL lysis buffer.</td></tr><tr><td>LDS (Lithium dodecyl sulfate)</td><td>Sigma Aldrich</td><td>L9781</td><td></td></tr><tr><td>15-mL ultrafiltration tube (10 kDa cutoff)</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>0.5-mL ultrafiltration tube (10 kDa cutoff)</td><td>Millipore</td><td>UFC501096</td><td></td></tr><tr><td>Streptavidin magnetic beads</td><td>Thermo Fisher Scientific</td><td>88816</td><td></td></tr><tr><td>DMSO (Dimethyl sulfoxide)</td><td>Sigma Aldrich</td><td>41639</td><td></td></tr><tr><td>Azide-biotin</td><td>Click Chemistry Tools</td><td>AZ104</td><td></td></tr><tr><td>Copper(II) sulfate</td><td>Sigma Aldrich</td><td>C1297</td><td></td></tr><tr><td>THPTA [Tris(3-hydroxypropyltriazolylmethyl)amine]</td><td>Sigma Aldrich</td><td>762342</td><td></td></tr><tr><td>Sodium ascorbate</td><td>Sigma Aldrich</td><td>11140</td><td></td></tr><tr><td>Azide-Cy5</td><td>Click Chemistry Tools</td><td>AZ118</td><td></td></tr><tr><td>LDS sample buffer (4&amp;times;)</td><td>Thermo Fisher Scientific</td><td>NP0008</td><td></td></tr><tr><td>10% bis-Tris gel</td><td>Thermo Fisher Scientific</td><td>NP0301BOX</td><td></td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>AM9260G</td><td></td></tr><tr><td>RNase A</td><td>Sigma Aldrich</td><td>R6513</td><td></td></tr><tr><td>SDS (Sodium dodecyl sulfate)</td><td>Thermo Fisher Scientific</td><td>15525017</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S3014</td><td></td></tr><tr><td>Brij-97 [Polyoxyethylene (20) oleyl ether]</td><td>J&amp;amp;K</td><td>315442</td><td></td></tr><tr><td>Triethanolamine</td><td>Sigma Aldrich</td><td>V900257</td><td></td></tr><tr><td>Streptavidin agarose</td><td>Thermo Fisher Scientific</td><td>20353</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U5378</td><td></td></tr><tr><td>Sarkosyl (&lt;em&gt;N&lt;/em&gt;-Lauroylsarcosine sodium salt)</td><td>Sigma Aldrich</td><td>61743</td><td></td></tr><tr><td>Biotin</td><td>Sigma Aldrich</td><td>B4501</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>30970</td><td></td></tr><tr><td>MaxQuant</td><td></td><td></td><td>Version: 1.5.5.1</td></tr></tbody></table>

capture and identification of rna-binding proteins by using click chemistry-assisted rna-interactome capture (caric) strategy

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

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 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 ...

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 ...

capture-identification-rna-binding-proteins-using-click-chemistry

Research

JoVE Journal

Biochemistry

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

This video demonstrates an in vitro RNA pull-down assay to identify RNA-binding proteins (RBPs), which interact with the adenylate-uridylate-rich element (ARE) sequences in mRNA. The target RBPs from a cell lysate are mixed with an RNA probe to form RNA-protein complexes. The complexes are isolated via affinity purification utilizing the affinity of the desthiobiotin label of the RNA probe to a streptavidin-labeled magnetic bead.

RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction

In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Learn the CARIC method to identify RNA-binding proteins in HeLa cells using click chemistry and UV cross-linking. This protocol captures proteins bound to coding and noncoding RNAs for post-transcriptional gene regulation studies.

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

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