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>
		<tr>
			<td>2 mM DTT</td>
		</tr>
		<tr>
			<td>1x protease inhibitor cocktail EDTA-free</td>
		</tr>
		<tr>
			<td>RNase Out 5 &#181;l/ml (RNase inhibitor)</td>
		</tr>
		<tr>
			<td>Cytoplasmic Lysis Buffer:</td>
		</tr>
		<tr>
			<td>0.2 M Sucrose</td>
		</tr>
		<tr>
			<td>1.2% Triton X-100</td>
		</tr>
		<tr>
			<td>NETN-150 Wash Buffer:</td>
		</tr>
		<tr>
			<td>20 mM Tris-HCl, pH 7.4</td>
		</tr>
		<tr>
			<td>150 mM NaCl</td>
		</tr>
		<tr>
			<td>0.5% NP-40</td>
		</tr>
		<tr>
			<td>3 mM MgCl2</td>
		</tr>
		<tr>
			<td>10% Glycerol</td>
		</tr>
		<tr>
			<td>Binding Buffer:</td>
		</tr>
		<tr>
			<td>10 mM HEPES pH 7.6&#160;</td>
		</tr>
		<tr>
			<td>40 mM KCl&#160;</td>
		</tr>
		<tr>
			<td>3 mM MgCl2</td>
		</tr>
		<tr>
			<td>2 mM DTT</td>
		</tr>
		<tr>
			<td>5% glycerol</td>
		</tr>
	</tbody>
</table>
Table 2: Buffer compositions.
1. Preparation of the Cells and the Affinity Matrix
<ol>
	<li>Culture a cell line of interest to sub-confluence (80%) in a 10 cm dish. Use 10 cm plates for independent immunoprecipitations (IP) of a FLAG-tagged NLS-MS2 coat protein. Perform the IP using antisera to the FLAG-epitope tag. When expressing the FLAG-tagged MS2 coat protein plasmid, transfect cells 24-48 hr in advance of harvest20. 
	NOTE: A particular RNP may be enriched from nuclear or cytoplasmic lysates or a biochemically-fractionated preparation, such as fractions of a sucrose gradient. It is recommended to harvest the lysate from non-transfected cells in parallel to institute an additional negative control.</li>
	<li>Transfer 60 &#181;l per IP of protein G magnetic bead slurry to a 1.7 ml microcentrifuge tube.</li>
	<li>Place the tube on a magnet rack for microcentrifuge tubes to separate the beads from the storage solution.</li>
	<li>Remove the storage solution by carefully drawing it up with a micropipette.</li>
	<li>Remove the tube from the magnet rack.</li>
	<li>Wash and equilibrate the beads with 600 &#181;l (10 times the used volume of beads) of 1x wash buffer (20 mM Tris-HCl, pH 7.4; 3 mM MgCl2; and 150 mM NaCl) and end-over-end rotation for 3 min at room temperature.</li>
	<li>Place the tube on the magnet rack and remove the wash buffer.</li>
	<li>Add 10 volumes (600 &#181;l) of 1x wash buffer and immunoprecipitating FLAG antibody to the equilibrated protein G magnetic beads (according to the amount recommended by the manufacturer for an immunoprecipitation) and rotate end-over-end at room temperature for at least 30 min to conjugate the immunoprecipitating antibody. Use the corresponding isotype IgG as a suitable antibody negative control.</li>
	<li>Place the tube on the magnet rack to collect the beads and to remove the supernatant.</li>
	<li>Remove the tube from the magnet rack and wash the antibody-conjugated beads with 10 volumes (600 &#181;l) of 1x wash buffer and rotation for 3 min at room temperature. Repeat this step twice.</li>
	<li>Place the tube on the magnet rack and remove the wash buffer.</li>
</ol>
2. Harvesting the RNPs
NOTE: Prepare the RNPs during the incubation time after step 1.8.
<ol>
	<li>Remove culture medium from the cells by aspiration and wash the cells twice with 1-5 ml of ice-cold 1x phosphate-buffered saline (PBS). Use a cell scraper to dislodge wet, adherent cells prior to collection by centrifugation at 226 x g for 4 min at 4 &#176;C.</li>
	<li>Add 375 &#181;l of ice-cold, low-salt buffer (20 mM Tris-HCl, pH 7.5; 3 mM MgCl2; 10 mM NaCl; 2 mM DTT; 1x protease-inhibitor cocktail, EDTA-free; and 5 &#181;l/ml RNase Inhibitor) to the cell pellet and allow swelling by placing it on ice for 5 min. 
	NOTE: Tailor the volume of low-salt buffer according the size of the cell pellet. For 1.2 x 106 cells from a 10 cm plate, 375 &#181;l of buffer is sufficient.</li>
	<li>To collect the cytoplasmic cell lysate, add 125 &#181;l of ice-cold lysis buffer (0.2 M sucrose/1.2% Triton X-100), and then perform 10 strokes with a Dounce homogenizer that was pre-chilled in an ice bucket. 
	NOTE: To collect the total cell extract, standard RIPA lysis buffer is recommended for solubilizing the nucleoplasm/chromatin.</li>
	<li>Spin in a microfuge at top speed (16,000 x g) for 1 min; this will clear the supernatant of debris and nuclei.</li>
	<li>Transfer the supernatant to a new 1.7 ml microcentrifuge tube that has been on ice. Determine the total protein concentration by a standard, laboratory-preferred method, such as the Bradford Assay23. Reserve an aliquot of at least 10% for a Western blot analysis, to be used as an input control. Use the harvested cell lysate immediately for immunoprecipitation or store it at -80 &#176;C for future analysis. 
	NOTE: In our experience, these samples can be stored and used several months later for immunoprecipitation analysis without a compromise of integrity.</li>
</ol>
3. Immunoprecipitation
<ol>
	<li>Add the desired volume of the harvested cell lysate, based upon the protein concentration determined in step 2.5, to the target antibody-conjugated beads. Using 1x wash buffer, bring the total volume up to 600 &#181;l and rotate end-over-end for 90 min at room temperature. 
	NOTE: This time period is sufficient to generate a robust isolation of high-affinity RNP complexes while minimizing non-specific binding. Dilute alternative RNP sources, such as fractions of a sucrose gradient, at least 1:1 with 1x wash buffer, and then incubate them with previously-prepared bead-antibody complexes, as mentioned above.</li>
	<li>After 90 min of incubation, place the IP tube on the magnet rack to collect the beads. Reserve the supernatant as flow-through. 
	NOTE: This first flow-through is an important control sample to measure IP efficiency. An immunoblot assay will provide an indication of IP efficiency, as well as the specificity of the investigated interactions. It is recommended to keep this supernatant for downstream analysis.</li>
	<li>Wash the RNP-bound, antibody-conjugated beads with 10 volumes (600 &#181;l) of ice-cold NETN-150 wash buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 3 mM MgCl2; 0.5% NP40; and 10% glycerol) under rotation for 3 min at room temperature. Repeat the step 3 times. 
	NOTE: This buffer differs from the lysis buffer and effectively reduces weak or non-specific associations.</li>
	<li>Following the final wash, resuspend the immobilized RNP complexes in 60 &#181;l of ice-cold 1x binding buffer (10 mM HEPES, pH 7.6; 40 mM KCl; 3 mM MgCl2; 5% glycerol; and 2 mM DTT) and reserve 10% of the immobilized complex beads for Western blot and 20% for RNA isolation.</li>
</ol>
4. Elution
<ol>
	<li>Adjust the remaining volume to 100 &#181;l with ice-cold 1x binding buffer and heat the tube at 70 &#176;C for 3 min.</li>
	<li>To isolate cognate RNA-protein complexes, add a ~30-nt DNA oligonucleotide that is complementary to the 3&#39; sequence boundary of the RNA of interest and incubate for 30 min at room temperature with gentle rocking (100 nM of oligonucleotide is sufficient). 
	NOTE: The oligonucleotide is antisense to the nucleotide residues adjacent to the translation start-site of the PCE construct used as an example in this protocol. Appropriate sequence complementarity is provided by the ~40% G-C content. Design the antisense oligonucleotide for efficient hybridization to the minimum complementary region of the target RNA.</li>
	<li>Add 5-10 units of RNase H to the tube and incubate at room temperature for 1 hr to cleave the RNA of the RNA:DNA hybrid. Transfer the supernatant to a sterile 1.7 ml microcentrifuge tube; this sample contains the captured RNP complexes of interest. 
	NOTE: Depending upon the accessibility of the cognate RNA, two or more rounds of RNase H treatment may be useful to increase the sample abundance for the downstream analyses.</li>
	<li>Use 20% of the eluate in a Western blot for known associated proteins and 20% of the eluate for RNA isolation followed by RT qPCR. The remaining 60% can be used for mass spectrometry or to identify protein components.</li>
	<li>In parallel, subject an aliquot of the reserved cell lysate to RNA isolation and RT-PCR analysis. This assessment provides an indication of the enrichment of RNA within an RNP complex. 
	NOTE: Use the isolated protein preparation in a control western blot to ascertain that the RNase H cleavage was effective in releasing the RNP from the immunuoprecipitated complex.</li>
</ol>
5. Protein Electrophoresis and Western Blot Analysis
<ol>
	<li>Subject approximately 10-20% of the total sample to SDS-PAGE and immunoblot analysis according to standard laboratory protocol. 
	NOTE: This step serves to validate IP efficiency and specificity prior to downstream RNA analysis. It can also be used to assess the protein composition of the isolated RNP complex.</li>
</ol>
6. Collection of the Immunoprecipitated RNA
NOTE: RNA isolation can be done by Trizol method or following the described protocol.
<ol>
	<li>Resuspend half of the total sample in 750 &#181;l of acid guanidinium thiocyanate reagent and incubate at room temperature for 5 min prior to extracting the RNA from the captured PCE-RNP complexes.</li>
	<li>Add 200 &#181;l of of chloroform to the tube, shake vigorously for 10 sec, and incubate at room temperature for 3 min.</li>
	<li>After centrifugation at 16,000 x g for 15 min at 4 &#176;C, collect the aqueous phase into afresh tube and add an equal volume of isopropanol. Mix well and incubate at room temperature for at least 10 min. Add 1 &#181;l of glycol blue to the sample and store it at -20 &#176;C in the freezer for efficient precipitation or processing at a future date.</li>
	<li>Centrifuge the tube at 16,000 x g for 10 min at 4 &#176;C. Carefully collect and discard the supernatant so as to not disturb the RNA pellet; the use of a p-200 micropipette tip is recommended to facilitate this process.</li>
	<li>Add 500 &#181;l of 75% ethanol to each tube, vortex, and centrifuge the tubes at 16,000 x g for 5 min at 4 &#176;C. Carefully collect and discard the supernatant as in step 6.4. Air-dry the pellet for 2-3 min and resuspend in 100 &#181;l of RNase-free water. 
	NOTE: Do not prolong the time the pellet air-dries in order to avoid a problem with resuspension.</li>
	<li>Apply 100 &#181;l of RNA sample to an RNA clean-up column. Process it using the manufacturer&#39;s protocol. Elute the RNA in 30 &#181;l of RNase-free water and store at -80 &#176;C for up to 3 months. 
	NOTE: This isolated RNA is suitable for downstream analysis by RT-realtime PCR (qPCR), microarray, and RNA sequencing. Depending upon the abundance of the RNP and the efficiency with which the RNP of interest is isolated, the same lysate may be subjected to two or more rounds of IP to generate sufficient RNA applicable for the downstream analyses.</li>
</ol>
7. RNA Reverse Transcription and Amplification of cDNA by PCR
<ol>
	<li>Subject the isolated RNA to reverse transcription by a random primer with a high-quality reverse transcriptase (RT), according to the manufacturer&#39;s instructions.</li>
	<li>To amplify the RNA of interest, design a gene-specific antisense primer within the RNase H cleavage sequence. Use similar amounts of the antisense oligonucleotide, a random primer, or an oligo-dT primer (See Materials Table). 
	NOTE: The oligo-dT primer and poly-adenylated mRNA provide positive control reactions for the RT-PCR reactions.</li>
	<li>Reserve 5% of the RT reaction (1 &#181;l) for a preparative qPCR done in tandem with negative-control lysate IP and positive-control DNA samples to define the cutoff and produce a standard curve. If the CT value of the preparative qPCR is beyond the range of the standard curve, dilute the RT reaction in consecutive 1:5 dilutions and repeat step 7.2. 
	NOTE: For an RNA sequencing and mass spectrometry technique, please see the Supplementary Method file. <a href="http://cloudfront.jove.com/files/ftp_upload/54391/Supplementary_method_File_Jove.docx" target="_blank">Please click here to view this Supplementary Method file. (Right-click to download.)</a></li>
</ol>

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The authors have nothing to disclose.

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. ...

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JoVE Journal

Biochemistry

8.9K Views.  University of Minnesota. 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. 

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

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 ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

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 ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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 ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

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 ...