Name: isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution
Uploaded: 2017-01-16T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

The authors gratefully acknowledge support by NIH P50GM103297, P30CA100730 and Comprehensive Cancer P01CA16058.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer Compositions</td>
		</tr>
		<tr>
			<td>Wash Buffer:</td>
		</tr>
		<tr>
			<td>50 mM Tris-HCl, pH 7.4</td>
		</tr>
		<tr>
			<td>150 mM NaCl</td>
		</tr>
		<tr>
			<td>3 mM MgCl2</td>
		</tr>
		<tr>
			<td>Low Salt Buffer:</td>
		</tr>
		<tr>
			<td>20 mM Tris-HCl, pH 7.5</td>
		</tr>
		<tr>
			<td>10 mM NaCl</td>
		</tr>
		<tr>
			<td>3 mM MgCl2</td>
		</tr>
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			<td>2...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat.Rev.Genet. 8 (7), 533-543 (2007).</li><li>Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+birth+to+death:+the+complex+lives+of+eukaryotic+mRNAs.">From birth to death: the complex lives of eukaryotic mRNAs.</a> Science. 309 (5740), 1514-1518 (2005).</li><li>Hassan, M. Q., Gordon, J. A., Lian, J. B., van Wijnen, A. J., Stein, J. L., Stein, G. 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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+RNA-protein+interactions+in+vivo+by+RNA+immunoprecipitation.">Studying RNA-protein interactions in vivo by RNA immunoprecipitation.</a> Methods Mol.Biol. 791, 253-264 (2011).</li><li>Singh, D., Boeras, I., Singh, G., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Cognate+Cellular+and+Viral+Ribonucleoprotein+Complexes+of+HIV-1+RNA+Applicable+to+Proteomic+Discovery+and+Molecular+Investigations.">Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.</a> Methods Mol.Biol. 1354, 133-146 (2016).</li><li>Stake, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+and+two+avian+retroviral+5'+untranslated+regions+bind+orthologous+human+and+chicken+RNA+binding+proteins.">HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.</a> Virology. 486, 307-320 (2015).</li><li>Sung, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analysis+using+microarray+identified+complex+signaling+pathways+modulated+by+hypoxia+in+nasopharyngeal+carcinoma.">Genome-wide expression analysis using microarray identified complex signaling pathways modulated by hypoxia in nasopharyngeal carcinoma.</a> Cancer Lett. 253 (1), 74-88 (2007).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat.Rev.Genet. 10 (1), 57-63 (2009).</li><li>Michlewski, G., Caceres, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNase-assisted+RNA+chromatography.">RNase-assisted RNA chromatography.</a> RNA. 16 (8), 1673-1678 (2010).</li><li>Tacheny, A., Dieu, M., Arnould, T., Renard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+identification+of+proteins+interacting+with+nucleic+acids.">Mass spectrometry-based identification of proteins interacting with nucleic acids.</a> J. Proteomics. 94, 89-109 (2013).</li><li>Duan, R., Sharma, S., Xia, Q., Garber, K., Jin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+RNA-mediated+neurological+disorders.">Towards understanding RNA-mediated neurological disorders.</a> J.Genet.Genomics. 41 (9), 473-484 (2014).</li><li>Butsch, M., Hull, S., Wang, Y., Roberts, T. M., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5'+RNA+terminus+of+spleen+necrosis+virus+contains+a+novel+posttranscriptional+control+element+that+facilitates+human+immunodeficiency+virus+Rev/RRE-independent+Gag+production.">The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE-independent Gag production.</a> J. Virol. 73 (6), 4847-4855 (1999).</li><li>Bolinger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+A+interacts+with+divergent+lymphotropic+retroviruses+and+promotes+translation+of+human+T-cell+leukemia+virus+type+1.">RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1.</a> Nucleic Acids Res. 35 (8), 2629-2642 (2007).</li><li>Bolinger, C., Sharma, A., Singh, D., Yu, L., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+A+modulates+translation+of+HIV-1+and+infectivity+of+progeny+virions.">RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions.</a> Nucleic Acids Res. 38 (5), 1686-1696 (2010).</li><li>Lee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+the+DHX9+helicase+induces+premature+senescence+in+human+diploid+fibroblasts+in+a+p53-dependent+manner.">Suppression of the DHX9 helicase induces premature senescence in human diploid fibroblasts in a p53-dependent manner.</a> J.Biol.Chem. 289 (33), 22798-22814 (2014).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72, 248-254 (1976).</li><li>Glenn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+protein+samples+for+mass+spectrometry+and+N-terminal+sequencing.">Preparation of protein samples for mass spectrometry and N-terminal sequencing.</a> Methods Enzymol. 536, 27-44 (2014).</li><li>Keene, J. D., Komisarow, J. M., Friedersdorf, M. 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The RNP isolation and cognate RNA identification strategy described here is a selective means of investigating a specific RNA-protein interaction and of discovering candidate proteins co-regulating a specific RNP in cells.
The advantage of using oligonucleotide-directed RNase H cleavage to isolate RNPs is the ability to capture and specifically analyze the RNP cis-acting RNA element over heterogeneous RNPs bound downstream to the cis-acting RNA element of interest. Because the abundance of a c...

Post-transcriptional gene expression is precisely regulated, beginning with DNA transcription in the nucleus. Controlled by RNA binding proteins (RBPs), mRNA biogenesis and metabolism occur in highly dynamic ribonucleoprotein particles (RNPs), which associate and dissociate with a substrate precursor mRNA during the progression of RNA metabolism1-3. Dynamic changes in RNP components affect the post-transcriptional fate of an mRNA and provide quality assurance during the processing of primary transcripts, their nuclear trafficking and localization, their activity as mRNA templates for translation, and the eventual turnover of mature mRNAs.
Numerous proteins are designated as RBPs by virtue of their conserved amino acid domains, including the RNA recognition motif (RRM), the double-stranded RNA binding domain (RBD), and stretches of basic residues (e.g., arginine, lysine, and glycine)4. RBPs are routinely isolated by immunoprecipitation strategies and are screened to identify their cognate RNAs. Some RBPs co-regulate pre-mRNAs that are functionally-related, designated as RNA regulons5-8. These RBPs, their cognate mRNAs, and sometimes non-coding RNA, form catalytic RNPs that vary in composition; their uniqueness is due to various combinations of associated factors, as well as to the temporal sequence, location, and duration of their interactions9.
RNA immunoprecipitation (RIP) is a powerful technique to isolate RNPs from cells and to identify associated transcripts using sequence analysis10-13. Moving from candidate to genome-wide screening is feasible through RIP combined with a microarray analysis14 or high-throughput sequencing (RNAseq)15. Likewise, co-precipitating proteins may be identified by mass spectrometry, if they are sufficiently abundant and separable from the co-precipitating antibody16,17. Here, we address the methodology for isolating RNP components of a specific cognate RNA from cultured human cells, although the approach is alterable for soluble lysates of plant cells, fungi, viruses, and bacteria. Downstream analyses of the material include candidate identification and validation by immunoblot, mass spectrometry, biochemical enzymatic assay, RT-qPCR, microarray, and RNAseq, as summarized in Figure 1.
Given the fundamental role of RNPs in controlling gene expression at the post-transcriptional level, alterations in the expression of component RBPs or their accessibility to cognate RNAs can be detrimental for the cell and are associated with several types of disorders, including neurological disease18. DHX9/RNA helicase A (RHA) is necessary for the translation of selected mRNAs of cellular and retroviral origins6. These cognate RNAs exhibit structurally-related cis-acting elements within their 5&#39; UTR, which is designated as the post-transcriptional control element (PCE)19. RHA-PCE activity is necessary for the efficient cap-dependent translation of many retroviruses, including HIV-1, and of growth regulatory genes, including junD6,20,21. Encoded by an essential gene (dhx9), RHA is essential to cell proliferation and its down-regulation eliminates cell viability22. The molecular analysis of RHA-PCE RNPs is an essential step to understanding why RHA-PCE activity is necessary to control cell proliferation.
The precise characterization of the RHA-PCE RNP components at steady state or upon physiological perturbation of the cell requires the selective enrichment and capture of the RHA-PCE RNPs in sufficient abundance for downstream analysis. Here, retroviral PCEgag RNA was tagged with 6 copies of the cis-acting RNA binding site for the MS2 coat protein (CP) within the open reading frame. The MS2 coat protein was exogenously co-expressed with PCEgag RNA by plasmid transfection to facilitate the RNP assembly in growing cells. RNPs containing the MS2 coat protein with cognate MS2-tagged PCEgag RNA were immunoprecipitated from the cell extract and captured on magnetic beads (Figure 2a). To selectively capture the RNP components bound to the PCE, the immobilized RNP was incubated with an oligonucleotide complementary to sequences distal to the PCE, forming an RNA-DNA hybrid that is the substrate for RNase H activity. Since PCE is positioned in the 5&#39; terminal of the 5&#39; untranslated region, the oligonucleotide was complementary to the RNA sequences adjacent to the retroviral translation start site (gag start codon). RNase H cleavage near the gag start codon released the 5&#39; UTR complex from the immobilized RNP, which was collected as the eluent. Thereafter, the sample was evaluated by RT-PCR to confirm the capture of PCEgag and by SDS PAGE and immunoblot to confirm the capture of the target MS2 coat protein. A validation of the PCE-associated RNA binding protein, DHX9/RNA helicase A, was then performed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- FLAG antibody</td>
			<td>Sigma</td>
			<td>F7425</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RHA antibody</td>
			<td>Vaxron</td>
			<td>PA-001</td>
			<td></td>
		</tr>
		<tr>
			<td>TRizol LS reagent</td>
			<td>Life technology</td>
			<td>10296-028</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>Ambion</td>
			<td>AM2292</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26184</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaeasy clean-up column</td>
			<td>Qiagen</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript reverse transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Out</td>
			<td>Invitrogen</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>98379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>17904</td>
			<td></td>
		</tr>
		<tr>
			<td>Random hexamer primers</td>
			<td>Invitrogen</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo-dT primers</td>
			<td>Invitrogen</td>
			<td>AM5730G</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>IDT</td>
			<td></td>
			<td>Gene specific primers for&#160; PCR amplification</td>
		</tr>
		<tr>
			<td>Oligonucleotide for RNase H mediated cleavage</td>
			<td>IDT</td>
			<td></td>
			<td>Anti-sense primer for target RNA</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco Life technology</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM tissue culture medium</td>
			<td>Gibco Life technology</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco Life technology</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S642-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-Rad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td></td>
			<td></td>
			<td>1.7 ml micro-centrifuge tube holding</td>
		</tr>
		<tr>
			<td>Laminar hood</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td>For animal tissue culture</td>
		</tr>
		<tr>
			<td>Protein gel apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Protein transfer apparatus</td>
			<td></td>
			<td></td>
			<td>Protein sample transfer</td>
		</tr>
		<tr>
			<td>Ready to use protein gels (4-15%)</td>
			<td></td>
			<td></td>
			<td>Protein sample separation</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td></td>
			<td></td>
			<td>Pellet down the sample</td>
		</tr>
		<tr>
			<td>Table top rotator</td>
			<td></td>
			<td></td>
			<td>Mix the sample end to end</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Mix the samples</td>
		</tr>
	</tbody>
</table>

isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

Prior RIP results identified retroviral gag RNAs and selected cellular RNAs that co-precipitate with DHX9/RHA, including HIV-16, junD6 and huR (Fritz and Boris-Lawrie, unpublished). The retroviral 5&#39; UTR has been demonstrated to co-precipitate with DHX9/RHA in the nucleus and to co-isolate in the cytoplasm on polyribosomes. It is uniquely defined as the cis-acting post-transcriptional control element (PCE)6. To isolate PCEgag RNA-RHA ribonucle...

Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

The overall goal of this RNA immunoprecipitation technique is to selectively isolate and characterize endogenous RNA protein complexes formed in eukaryotic cells via a specific oligonucleotide directed enrichment. This method can help answer key questions in the post-transcriptional control field, such as the composition of RNA binding proteins that define the five prime UTR of a retroviral transcript to regulate its gene expression. The main advantage of this technique is that it allows a researcher to selectively isolate and characterize a specific RNA protein complex of interest while maintaining the possibility to conduct a more global analysis of the RNP downstream.Well the implications of this technique extend toward fundamental understanding of viral infections and numerous cellular diseases. And provides significant insight into the mechanisms of post-transcriptional control of gene expression. That could mean new viable drug targets.After culturing cells and carrying out immunoprecipitation of a FLAG-tagged protein according to the text protocol, transfer 60 microliters per IP of pro-TG magnetic bead slurry to a 1.7 milliliter microcentrifuge tube. Place the tube on a magnetic rack and remove the storage solution by carefully pipetting. Then remove the tube from the rack.Add 600 microliters of 1x wash buffer. And place the tube on an end over end rotator at room temperature for three minutes, to wash and equilibrate the beads. Place the tube on the magnetic rack and remove the wash buffer.Then add 10 volumes of fresh 1x wash buffer. Add immunoprecipitating FLAG antibody to the equilibrated pro-TG magnetic beads. And then rotate the tube at room temperature for at least 30 minutes, to conjugate the immunoprecipitating antibody.Place the tube on the magnet and remove the supernatant. Then remove the tube from the magnet and add 1x wash buffer to the beads and rotate at room temperature for three minutes. Repeat the wash step twice and remove the final buffer.Remove the culture medium from the cells by aspiration and use 1.5 milliliters of ice-cold PBS to wash the cells twice. With a cell scraper dislodge the wet, adherent cells. Pipette the cell suspension into fresh tubes and then centrifuge the culture at 226 times G in four degrees celsius, for four minutes.Add 375 microliters of ice cold low salt buffer to the cell pellet, and incubate the sample on ice for five minutes to allow swelling. It is important to take care when adding and re-suspending the cell pellet in the low salt lysis buffer, to ensure complete submersion and swelling without premature mechanical disruption. To collect the cytoplasmic cell lysate, add 125 microliters of ice cold lysis buffer, and then use a pre-chilled Dounce homogenizer to perform ten strokes.Dounce homogenize with enough force to enable plasma membrane lysis and contaminants of the released cytoplasmic lysate. But take care to not overextend the mechanical disruption and risk nuclear contaminations, disruption of RNA protein complex, or loss of released Spin the homogenate in a microfuge at top speed for one minute. Then transfer the supernatant to a new 1.7 milliliter tube that has been on ice.Determine the total protein concentration. Reserve 10%for western blot analysis, to be used as input control. To carry out immunoprecipitation, add the desired volume of harvested cell lysate to the target antibody conjugated beads.Using 1x wash buffer, bring the total volume up to 600 microliters and rotate the sample at room temperature. After 90 minutes of incubation, place the IP tube on the magnet rack to collect the beads. Then collect the supernatant and reserve it as flow through.Next, add 10 volumes of ice cold NETN 150 wash buffer to the RNP bound, antibody conjugated beads, and rotate the sample at room temperature for three minutes. After re-suspending the immobilized RNP complexes in 1x binding buffer according to the text protocol, adjust the remaining volume in 100 microliters of ice cold 1x binding buffer. And heat the tube at 70 degrees Celsius for three minutes.To isolate cognate RNA protein complexes, add an approximately 30 nucleotide DNA oligo, complementary to the three prime sequence boundary of the RNA of interest. And incubate for 30 minutes at room temperature with gentle rocking. Add five to ten units or RNase H to the tube and incubate the sample at room temperature for one hour to cleave the RNA from the RNA DNA hybrid.Then transfer the supernatant containing the captured RNP complexes of interest to a sterile 1.7 milliliter microcentrifuge tube. To collect the immunoprecipitated RNA, re-suspend half of the total sample of captured RNA complexes in 750 microliters of acid guanidinium thiocyanate reagent. Add 200 microliters of chloroform to the tube, shake vigorously for 10 seconds an incubate at room temperature for three minutes.After centrifugation, collect the aqueous phase in a fresh tube, and add an equal volume of isopropanol. Mix well and incubate the sample at room temperature for at least ten minutes. Add one microliter of glycol blue to the sample and store it at negative 20 degrees celsius for efficient precipitation or processing at a future date.Centrifuge the tube at 16, 000 times G in four degrees Celsius for ten minutes. The use a P200 micropipette to carefully collect and discard the supernatant so as not to disturb the RNA pellet. Add 500 microliters of 75%ethanol to each tube.Vortex and centrifuge the tubes for five minutes. Carefully collect and discard the supernatant as just demonstrated. Air dry the pellet for two to three minutes and use 100 microliters of RNase free water to re-suspend it.Apply 100 microliters of the RNA sample to an RNA clean up column. And process it according to the manufacturers protocol. Finally, elute the RNA in 30 microliters of RNase free water and store it at negative 80 degrees Celsius for up to three months.To isolate post-transcriptional control elements, or PCE, GAG, RNA, RHA ribonucleoprotein complexes formed in proliferating cells. FLAG-MS2 RNP immunoprecipitation was carried out in transfected HECK293 cell lysates. The results show efficient precipitation of the FLAG-MS2 protein.As DHX9/RHA is found in the RNP complex and not within the IgG isotope control, nor within the negative control. Shown here are western blots from RNase H digested RNA DNA complexes. RNase H cleaved PCE GAG RNA, releasing the RNP complex specifically bound to the five prime UTR.The DHX9/RHA associated RNPs were enriched in the eluents. Importantly, the FLAG-MS2 bound RNPs remained with the antibody conjugated beads. In this experiment, HO immunoprecipitation of the MS2 stem loop, containing retroviral PCE GAG RNA in cells and in eluents, was validated by RT-PCR and QRT-PCR.The results confirmed a select association between DHX9/RHA and the retroviral five prime UTR, which has been highlighted as a unique RNP, important for targeted translation control. Once mastered, this technique can be done in approximately five hours, if it is performed properly. While attempting this procedure, it's important to remember to perform all steps efficiently and with care, to ensure maintenance of intact RNP complexes and the isolation of the specific RNP complex of interest.Following this procedure, other methods like mass spectrometry and RNA-Seq, can be performed in order to answer additional questions, like global protein association with the target RNA. And region or complete analysis of all transcripts engaged within a particular RNP. After its development, this technique paved the way for researcher in the field of post-transcriptional gene control to explore the distinct RNA protein complexes governing pathogenic viral gene expiration and general mechanism of cellular gene expression control.After watching this video, you should have a good understanding of how to harvest intact cellular RNP complexes and determine their composition using selective oligonucleotide directed enrichment. Do not forget too, working with pathogenic viruses and biological samples and hazardous chemicals like acid or phenol, can be extremely hazardous. All precautions, such as appropriate personal protective equipment should always be taken while performing this procedure.

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Research

JoVE Journal

Biochemistry

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...