Name: transcriptome-wide profiling of protein-rna interactions by cross-linking and immunoprecipitation mediated by flag-biotin tandem purification
Uploaded: 2020-05-18T13:00:00
Description: Watch this Scientific Journal Video about Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification at JoVE.com

Grant support is from the National Basic Research Program of China (2017YFA0504204, 2018YFA0107604), the National Natural Science Foundation of China (31630095), and the Center for Life Sciences at Tsinghua University.

1. Cell line construction
<ol>
	<li>Clone the gene of interest into a PiggyBac vector pPiggyBac-FLAG-bio-[cDNA of interest]-(Hygromycin-resistant) plasmid that carries a FLAG-biotin epitope13,21 to express a FLAG-biotin tag fused RBP (FBRBP).</li>
	<li>Cotransfect the FBRBP expressing vector with the pBase vector into a cell line expressing the BirA enzyme22. 
	NOTE: In this study, the FBRBP gene was...

<ol>
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Here we introduce a modified CLIP-seq method called FbioCLIP-seq, taking advantage of the FLAG-biotin double tagging system to perform tandem purification of protein-RNA complexes. The FLAG-biotin double tagging system has been shown to be powerful in identifying protein-protein and protein-DNA interactions13,21. Here we demonstrate the high specificity and convenience of this system in identifying the RNAs interacting with proteins. Through tandem pur...

RNAs and RNA-binding proteins (RBPs) control diverse cellular processes including splicing, translation, ribosome biogenesis, epigenetic regulation, and cell fate transition1,2,3,4,5,6. The delicate mechanisms of these processes depend on the unique spatial and temporal arrangement of RNAs and RBPs. Therefore, an important step towards understanding RNA regulation at the molecular level is to reveal the positional information about the binding sites of RBPs.
A strategy referred to as cross-linking and immunoprecipitation (CLIP-seq) has been developed to capture protein-RNA interactions with UV cross-linking followed by immunoprecipitation of the protein of interest7. The key feature of the methodology is the induction of covalent cross-links between an RNA-binding protein and its directly bound RNA molecules (within ~1 &#197;) by UV irradiation8. The RBP footprints can be determined by CLIP tag clustering and peak calling, which usually have a resolution of 30&#8722;60 nt. Alternatively, the reverse transcription step of CLIP can lead to indels (insertions or deletions) or substitutions to the cross-linking sites, which allows identification of protein binding sites on the RNAs at a single-nucleotide resolution. Pipelines like Novoalign and CIMS have been developed for the analysis of the high-throughput sequencing results of CLIP-seq8. Several modified CLIP-seq methods have also been proposed, including individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), enhanced CLIP (eCLIP), irCLIP, and photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP)9,10,11,12.
Despite the wide use of traditional CLIP-seq methods in the study of RBPs, the CLIP methods have several drawbacks. First, the tedious denatured gel electrophoresis and membrane transfer procedure may lead to loss of information, and cause limited sequence complexity. Second, the protein specific antibody-based CLIP method may pull down a protein complex instead of a single target protein, which may lead to false positive protein-RNA interactions from the copurified RBPs. Third, the antibody-based strategy requires a large amount of high-quality antibodies, which makes the application of these methods inadequate for the study of RBPs without high-quality antibodies available. Fourth, the traditional CLIP method requires radiolabeled ATP to label the protein-bound RNAs.
The high affinity of streptavidin to biotinylated proteins makes it a very powerful approach to purify specific proteins or protein complexes. The efficient biotinylation of proteins carrying an artificial peptide sequence by ectopically expressed bacterial BirA biotin ligase in mammalian cells makes it an efficient strategy to perform biotin purification in vivo13. We developed a modified CLIP-seq method called FbioCLIP-seq (FLAG-Biotin-mediated Cross-linking and Immunoprecipitation followed by high-throughput sequencing) using FLAG-biotin tag tandem purification14 (Figure 1). Through tandem purification and stringent wash conditions, almost all the interacting RBPs are removed (Figure 2). The stringent wash conditions also allow circumventing the SDS-PAGE and membrane transfer, which is labor intensive and technically challenging. And similar to eCLIP and irCLIP, the FbioCLIP-seq method is isotope-free. Skipping the gel running and transfer steps avoids the loss of information, keeps authentic protein-RNA interactions intact, and increases the library complexity. Moreover, the high efficiency of the tagging system makes it a good choice for RBPs without high-quality antibodies available.
Here we provide a step-by-step description of the FbioCLIP-seq protocol for mammalian cells. Briefly, cells are cross-linked by 254 nm UV, followed by cell lysis and FLAG immunoprecipitation (FLAG-IP). Next, the protein-RNA complexes are further purified by biotin affinity capture and RNAs are fragmented by partial digestion with MNase. Then, the protein-bound RNA is dephosphorylated and ligated with a 3&#8217; linker. A 5&#8217; RNA linker is added after the RNA is phosphorylated with PNK and eluted by proteinase K digestion. After reverse transcription, the protein-bound RNA signals are amplified by PCR and purified by agarose gel purification. Two RBPs were chosen to exemplify the FbioCLIP-seq result. LIN28 is a well-characterized RNA-binding protein involved in microRNA maturation, protein translation, and cell reprogramming15,16,17. WDR43 is a WD40 domain-containing protein thought to coordinate ribosome biogenesis, eukaryotic transcription, and embryonic stem cell pluripotency control14,18. Consistent with previously reported results for LIN28 with CLIP-seq, FbioCLIP-seq reveals binding sites of LIN28 on &#8220;GGAG&#8221; motifs in the microRNA mir-let7g and mRNAs16,19 (Figure 3). WDR43 FbioCLIP-seq also identified the binding preference of WDR43 with 5&#39; external transcribed spacers (5&#39;-ETS) of pre-rRNAs20 (Figure 4). These results validate the reliability of the FbioCLIP-seq method.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>UVP</td>
			<td>HL-2000 HybrilLinker</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Affinity Purification Beads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG beads</td>
			<td>Sigma-Aldrich</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin beads</td>
			<td>Invitrogen</td>
			<td>112.06D</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco</td>
			<td>70013032</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M NaOAc</td>
			<td>Ambion</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>3 x FLAG peptide</td>
			<td>Sigma-Aldrich</td>
			<td>F4799</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>CIP</td>
			<td>NEB</td>
			<td>M0290S</td>
			<td>CIP buffer is in the same package.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel purification kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Ambion</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>449172</td>
			<td></td>
		</tr>
		<tr>
			<td>MNase</td>
			<td>NEB</td>
			<td>M0247S</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Amresco</td>
			<td>M158-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Porteinase K</td>
			<td>TAKARA</td>
			<td>9033</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity 2X Master Mix</td>
			<td>NEB</td>
			<td>0492S</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse trancriptase (SupperScriptIII)</td>
			<td>Invitrogen</td>
			<td>18080093</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation reagent (Trizol)</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>ThermoFisher</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Invitrogen</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>RQ1 Dnase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>1614363</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td>PNK buffer is in the same package.</td>
		</tr>
		<tr>
			<td>T4 RNA ligaes</td>
			<td>ThermoFisher</td>
			<td>EL0021</td>
			<td>T4 RNA ligase buffer and BSA are in the same package.</td>
		</tr>
		<tr>
			<td>T4 RNA ligase2, truncated</td>
			<td>NEB</td>
			<td>M0242S</td>
			<td>T4 RNA ligase buffer and 50% PEG are in the same package.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>208884</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mESC culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (80%)</td>
			<td>Gibco</td>
			<td>11965126</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS (15%)</td>
			<td>Hyclone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (1%)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>LIF</td>
			<td></td>
			<td></td>
			<td>purified recombinant protein; 10,000 fold dilution</td>
		</tr>
		<tr>
			<td>NEAA (1%)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleoside mix (1%)</td>
			<td>Millipore</td>
			<td>ES-008-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (1%)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
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			<td></td>
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			<td>Kit</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>DNA gel extraction kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
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transcriptome-wide profiling of protein-rna interactions by cross-linking and immunoprecipitation mediated by flag-biotin tandem purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

The schematic representation of the FbioCLIP-seq procedure is shown in Figure 1. Compared with FLAG-mediated or streptavidin-mediated one-step affinity purification, FLAG-biotin tandem purification removed almost all the copurified proteins, avoiding the contamination of indirect protein-RNA interactions (Figure 2). Representative results for FbioCLIP-seq for LIN28 and WDR43 are depicted in Figure 3 and <strong...

Watch this Scientific Journal Video about Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification at JoVE.com

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

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.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

Only and only binding proteins control multiple biological processes. This FLAG-Biotin CLIP-seq method, hence to globally profile the special and the temporal arrangement of RNAs and RBPs in mammalian cells. This method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and the membrane transfer procedures.Compared to traditional CLIP-seq is isotope free and it doesn't require protein specific antibody. Begin by plating about 3 million mouse embryonic STEM cells in a 10 centimeter plate and incubating them overnight. On the next day, remove the medium with a vacuum and treat the cells with two milliliters of 0.25%trypsin EDTA for two minutes at room temperature.Quench the trypsin with four milliliters of fresh MESC medium, and transfer the cells to a 15 milliliter centrifuge tube. Then, centrifuge them at 300 times G for three minutes at four degrees Celsius and remove the supernatant. Suspend the cells in four milliliters of cold PBS and transfer them back to the 10 centimeter plate for cross-linking.Radiate the cells with a 254 nanometer UV light, then transfer the cross-linked cells to a 15 milliliter tube. Spin down this cells at 300 times G for three minutes at four degrees Celsius, then remove the supernatant and lyse the cells with 500 microliters of Buffer A prepared according to manuscript directions. Transfer the cells to an RNAse-free 1.5 milliliter tube, and incubate them at four degrees Celsius with gentle rotation for 30 to 60 minutes.Treat the lysate with 30 microliters of DNAse 1 at 37 degrees Celsius for 10 minutes. Then, stop the reaction by adding four microliters of 0.5 molar EDTA. Spin down the insoluble pellet by centrifuging at 12, 000 times G for 20 minutes at four degrees Celsius, and transfer the supernatant to a new pre chilled 1.5 milliliter tube.Transfer the cell lysate to pre equilibrated FLAG beads, and incubate the sample at four degrees Celsius while rotating for two to four hours. After the incubation, centrifuge the beads for two minutes at 3000 times G and remove the supernatant. Add 500 microliters of Buffer A to the sample, and incubated at 4 degrees Celsius for five minutes, then spin down the beads and remove the supernatant.Repeat the wash with Buffer A, followed by two washes with pre chilled Buffer B.To elute with a three X FLAG-peptide, prepare the elution solution as described in the text manuscript and spin down the FLAG-beads. Remove the supernatant with a narrow end pipette tip and add 200 microliters of the three X FLAG elution solution to each sample. Incubate the samples for 30 minutes at four degrees Celsius with gentle rotation.Then spin down the beads for two minutes at 3000 times G.Transfer the supernate to a fresh tube and repeat the elution twice. Save 5%of the eluents for Western blot analysis or silver staining to check the efficiency of FLAG immunoprecipitation. Transfer the pulled eluents to prepared streptavidin beads and rotate the samples gently at four degrees Celsius for one to three hours or overnight.Then, collect the beads on a magnetic stand and remove the supernatant. Wash the beads twice with 500 microliters of Buffer C with gentle rotation for five minutes per wash. Next, wash the beads twice with 500 microliters of Buffer D, and twice with 500 microliters of ice-cold PNK buffer, collecting the beads on the magnetic stand and removing the supernatant in between washes.To perform partial RNA digestion, add 100 microliters of MNA solution to each sample and vortex set at 37 degrees Celsius with a thermal mixer for 10 minutes. Collect the beads with a magnetic stand for 30 seconds and remove the supernatant. Then, wash them with PNK-EDTA buffer, Buffer C, and PNK buffer according to manuscript directions.Collect the beads with the magnetic stand and remove the buffer. Then, add the CIP reaction mix and vortex the beads at 37 degrees Celsius with a thermal mixer for 10 minutes. Quickly wash the beads twice with 500 microliters of ice-cold PNK EDTA buffer, followed by two washes with 500 microliters of ice-cold PNK buffer.After the washes, add 40 microliters of three prime linker ligation mix to the beads, and vortex them at 16 degrees Celsius with a thermal mixer for three hours or overnight. Efficient ligation of the linker to the allies is critical for this experiment. Check the reaction tube occasionally to make sure that these do not precipitate during ligation.After the incubation, repeat the washes with PNK EDTA and PNK buffers, add 40 microliters of PNK mixed to the beads, and vortex them at 37 degrees Celsius with the thermal mixer for 10 minutes. Wash the beads with PNK EDTA and PNK buffers, then proceed with RNA isolation as described in the text manuscript. Compared with FLAG-mediated or streptavidin mediated one step affinity purification, FLAG-Biotin tandem purification removed almost all the core purified proteins, avoiding the contamination of indirect protein RNA interactions.LIN28 and WDR43, Fbio CLIP-seq was performed using mouse embryonic STEM cells. In total, about 7.7 million and 2.2 million unique reads were retrieved for LIN28 and WDR43 respectively. Comparison of the track views show different binding patterns in the RN45 pre-rRNA locus.The cross-linked sites on the GGA G motif of pre-let7-g RNA by LIN28, Fbio CLIP-seq or identified. Furthermore, enriched RNA motifs in the binding sites, and the percentage of mutated nucleotides in different types of mutations were determined. Showing that LIN28 prefers to bind to the gene nucleotide.Well, at 10:00 PM, this protocol, it is important to confirm the efficiency of the immunoprecipitation to make sure that the protein only complexes were purified successfully.

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