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.

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
<ol>
	<li>Metabolic incorporation of EU and 4SU
	<ol>
		<li>Culture HeLa...

<ol>
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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...

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 border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HeLa</td>
			<td style="width:175px;">ATCC</td>
			<td style="width:175px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:351px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FBS (Fetal Bovine Serum)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Penicillin &#38; Streptomycin</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EU (5-ethynyl uridine)</td>
			<td style="width:175px;">Wuhu Huaren Co.</td>
			<td style="width:175px;">CAS:69075-42-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">4SU (4-thiouridine)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">T4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">10&#215;PBS (Phosphate-Buffered Saline)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">AM9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">UV cross-linker</td>
			<td style="width:175px;">UVP</td>
			<td style="width:175px;">CL-1000</td>
			<td>Equiped with 365-nm UV lamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DEPC (Diethyl pyrocarbonate)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">D5758</td>
			<td>To treat water. Highly toxic!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tris&#183;HCl, pH 7.5</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LiCl</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">62476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nonidet P-40</td>
			<td style="width:175px;">Biodee</td>
			<td style="width:175px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA-free protease inhibitor cocktail</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">88265</td>
			<td>One tablet for 50 mL lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LDS (Lithium dodecyl sulfate)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">L9781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">15-mL ultrafiltration tube (10 kDa cutoff)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:175px;">UFC901024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">0.5-mL ultrafiltration tube (10 kDa cutoff)</td>
			<td style="width:175px;">Millipore</td>
			<td style="width:175px;">UFC501096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin magnetic beads</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">88816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">41639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Azide-biotin</td>
			<td style="width:175px;">Click Chemistry Tools</td>
			<td style="width:175px;">AZ104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Copper(II) sulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">THPTA [Tris(3-hydroxypropyltriazolylmethyl)amine]</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">762342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium ascorbate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Azide-Cy5</td>
			<td style="width:175px;">Click Chemistry Tools</td>
			<td style="width:175px;">AZ118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LDS sample buffer (4&#215;)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">10% bis-Tris gel</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">NP0301BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">AM9260G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">RNase A</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">R6513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SDS (Sodium dodecyl sulfate)</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">15525017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NaCl</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Brij-97 [Polyoxyethylene (20) oleyl ether]</td>
			<td style="width:175px;">J&#38;K</td>
			<td style="width:175px;">315442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triethanolamine</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">V900257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin agarose</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:175px;">20353</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">U5378</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sarkosyl (N-Lauroylsarcosine sodium salt)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">61743</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Biotin</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">B4501</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium deoxycholate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:175px;">30970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">MaxQuant</td>
			<td style="width:175px;"></td>
			<td style="width:175px;"></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

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 ...

Watch this Scientific Journal Video about Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy at JoVE.com

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

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.

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 ...

This method, CARIC, can help answer key questions in the field of post-transcription of gene recreation by providing a comprehensive census of RNA-binding proteins. The main advantage of CARIC is its ability to capture proteins bound to various kinds of RNAs such as mRNAs and non-coding RNAs. This method can be used in various types and the organism to study the RNA protein interaction at work.First, culture HeLa cells in supplemented DMEM at 37 degrees Celsius in a five percent CO2 atmosphere. When the cells reach about 80 percent confluence replace the culture medium on each dish with 15 milliliters of prewarmed, fresh medium. Add 15 microliters of 100 millimolar EU to each dish to a final concentration of one millimolar.For experimental and no UV control samples add 7.5 microliters of 100 millimolar four SU to each dish. Cover the dishes with foil and culture the cells for 16 hours using the previous conditions. After this, add half of the previous amount of EU and/or four SU to the appropriate plates and continue culturing for another two hours.To begin in vivo cross-linking, remove the culture medium and wash each dish three times with PBS using five milliliters of PBS per wash. Remove the residual PBS as much as possible. For the experimental and no four SU control samples, place the dishes on ice with the lid removed.Use a UV cross-linker to irradiate the cells with UV light at 365 nanometers and at two jules per centimeter squared. For the no UV control samples, place the dishes on ice and protect them from light. Next, add one milliliter of prelysis buffer to each dish.Use a rubber cell lifter to scrape the cells and collect the prelysis suspension in a 15 milliliter tube. Add prelysis buffer to a tube containing the suspension from two dishes adjusting the total volume to six milliliters. Then add an equal volume of R lysis buffer.Pass the cell lysate through a syringe with a narrow needle several times until the lysate is clear and homogeneous. Incubate the lysate at four degrees Celsius with gentle rotation for one hour. After this dilute the lysate in 20 volumes of dilution buffer and divide it into 15 milliliter fractions in 15 milliliter untrafiltration tubes, molecular cutoff of 10 kilodaltons.Using a swinging bucket rotator, spin the tubes at 4000 times G and four degrees Celsius for about 15 minutes until each fraction is concentrated to a volume less than one milliliter. Add 14 milliliters of dilution buffer to each concentrated lysate fraction and repeat the concentration process. Then combine the fractions and concentrate them to a volume of six milliliters using the previously described concentration process.First prepare the reaction mix as outlined in the text protocol. Add this reaction mix to the six milliliters of pre-cleared lysate and mix well. Add 162.5 microliters of reducing reagent and mix well.Incubate at room temperature on an orbital shaker at 800 rpm for two hours. After this quench the reaction by adding five millimolar EDTA and incubating on the orbital shaker at room temperature for five minutes. Next, split the reaction mixture between two 50 milliliter tubes, each containing four volumes of pre-chilled methanol and incubate at negative 30 degrees Celsius for 30 minutes.Centrifuge at 4000 times G and four degrees Celsius for 15 minutes. Discard the supernatant and add between one and two milliliters of pre-chilled methanol to the pellet. Pipet up and down to break up the pellet making sure that the pellet ends up completely suspended with no visible chunks.Repeat the centrifugation and resuspension process twice. After this, carefully draw out the residual methanol as much as possible making sure not to disturb the pellet. Add 10 milliliters of reconstitution buffer and pipet up and down to dissolve the pellet.Centrifuge at 4000 times G and four degrees Celsius for 10 minutes. Then transfer the supernatant to a new tube making sure to collect 20 microliters of the sample for quality control. Transfer the cleaned up and reconstituted sample to the prepared streptavidin agarose beads.Incubate at four degrees Celsius with gentle rotation overnight. Spin down the beads at 4000 times G for five minutes. Transfer the supernatant to a new tube making sure to collect 20 microliters of the sample for quality control.Wash the beads with 10 milliliters of wash buffer A.Incubate with gentle rotation at 12 rpm and at room temperature for 10 minutes. Then spin down the beads at 4000 times G for five minutes. Remove the supernatant and repeat the washing and centrifugation process once more.Repeat this wash process for wash buffer B and wash buffer C as outlined in the text protocol. After this, wash the beads with 10 milliliters of 50 millimolar Tris hydrochloride. Spin the beads down at 4000 times G for five minutes.Remove the supernatant and split the beads evenly between two 1.5 microcentrifuge tubes. To begin eluding the captured RNPs, add 400 microliters of prepared biotin elution buffer to 400 microliters of washed, settled beads. Incubate on an orbital shaker at 1500 rpm at room temperature for 20 minutes.Then incubate on an orbital shaker with a heat block at 1500 rpm and 65 degrees Celsius for 10 minutes. Centrifuge the beads at 7800 times G for one minute and collect the eluded RNP. Add 400 microliters of fresh biotin elution buffer to the beads.Repeat the incubation and centrifugation process to collect a second elute and combine the two elutes in a single 15 milliliter tube. Next, add three volumes of dilution buffer to the combined RNP elutes to decrease the concentration of SDS. Using a 0.5 milliliter ultra filtration tube, concentrate the sample to about 40 microliters as outlined in the text protocol.Add 0.5 micrograms per microliter RNase A and incubate at 37 degrees Celsius for two hours to release RBPs from cross-linked RNase. Collect two microliters of RBPs for quality control. When optimizing CARIC protocols, quality control steps are critical.Representative quality control of the click labeled samples is performed via in-gel fluorescence analyses. Only the doubly labeled sample shows a strong smear band at the high molecular weight which represents the signal of cross-linked RNPs. The RNP signal is abolished by omitting either the four SU or EU.It can also be abolished by digestion with RNase A.In some cases a strong smeared band is observed in the no four SU control sample.This band represents the labeled uncross-linked RNase which will degrade during the heat innaturation in most cases. Quality control of the affinity poll dataficiency is assessed through western blot analysis which shows the biotin signals in the samples both before pulldown and after pulldown. A silver staining analysis of the captured RBPs reveals that for HeLa cells, the general total capture efficiency is between 0.05 and 0.1 percent of input proteins.While attempting this procedure it's important to remember that the RNase are sensitive to ribonucleases. Keep the experimental environment as clean as possible to reduce degredation of RNase. Following this procedure, protein almake identification using mass spectrometry can be preformed to reveal the iron-bounding proteins.After its development, the CARIC technique will pave the way for a more comprehensive understanding of the postal transcription OT regulation at work.

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

Research

JoVE Journal

Biochemistry

9.2K Views.  Peking University. Questo metodo, CARIC, può aiutare a rispondere a domande chiave nel campo della post-trascrizione della ricreazione genica fornendo un censimento completo delle proteine leganti l'RNA. Il principale vantaggio del CARIC è la sua capacità di catturare proteine legate a vari tipi di RNA come mRNA e RNA non codificanti. Questo metodo può essere utilizzato in vari tipi e nell'organismo per studiare l'interazione della proteina RNA sul lavoro.In primo luogo, coltura cellule HeLa in DMEM ...