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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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>

<ol>
	<li>Bandziulis, R. J., Swanson, M. S., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+as+developmental+regulators.">RNA-binding proteins as developmental regulators.</a> Genes Dev. 3 (4), 431-437 (1989).</li><li>Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+RNA-binding+proteins+interact+with+functionally+related+sets+of+RNAs,+suggesting+an+extensive+regulatory+system.">Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system.</a> PLoS Biol. 6 (10), e255 (2008).</li><li>Glisovic, T., Bachorik, J. L., Yong, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+and+post-transcriptional+gene+regulation.">RNA-binding proteins and post-transcriptional gene regulation.</a> FEBS Lett. 582 (14), 1977-1986 (2008).</li><li>Lunde, B. M., Moore, C., Varani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins:+modular+design+for+efficient+function.">RNA-binding proteins: modular design for efficient function.</a> Nat.Rev.Mol.Cell Biol. 8 (6), 479-490 (2007).</li><li>Cochrane, A. W., McNally, M. T., Mouland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retrovirus+RNA+trafficking+granule:+from+birth+to+maturity.">The retrovirus RNA trafficking granule: from birth to maturity.</a> Retrovirology. 3, 18 (2006).</li><li>Hartman, T. R., Qian, S., Bolinger, C., Fernandez, S., Schoenberg, D. R., 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+is+necessary+for+translation+of+selected+messenger+RNAs.">RNA helicase A is necessary for translation of selected messenger RNAs.</a> Nat.Struct.Mol.Biol. 13 (6), 509-516 (2006).</li><li>Pullmann, R., 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=Analysis+of+turnover+and+translation+regulatory+RNA-binding+protein+expression+through+binding+to+cognate+mRNAs.">Analysis of turnover and translation regulatory RNA-binding protein expression through binding to cognate mRNAs.</a> Mol.Cell.Biol. 27 (18), 6265-6278 (2007).</li><li>Keene, J. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleoprotein+immunoprecipitation+(RNP-IP):+a+direct+in+vivo+analysis+of+microRNA-targets.">Ribonucleoprotein immunoprecipitation (RNP-IP): a direct in vivo analysis of microRNA-targets.</a> J.Cell.Biochem. 110 (4), 817-822 (2010).</li><li>Selth, L. A., Close, P., Svejstrup, J. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP-Chip:+the+isolation+and+identification+of+mRNAs,+microRNAs+and+protein+components+of+ribonucleoprotein+complexes+from+cell+extracts.">RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts.</a> Nat.Protoc. 1 (1), 302-307 (2006).</li><li>Ranji, A., Shkriabai, N., Kvaratskhelia, M., Musier-Forsyth, K., 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=Features+of+double-stranded+RNA-binding+domains+of+RNA+helicase+A+are+necessary+for+selective+recognition+and+translation+of+complex+mRNAs.">Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs.</a> J.Biol.Chem. 286 (7), 5328-5337 (2011).</li></ol>

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 cognate RNA in a given RNP is a minor fraction of the collected RNPs isolated without RNase H cleavage, the major disadvantage to this workflow is the scarcity of the cognate RNP. In our experience, this limitation necessitated 5- to 10-fold more starting material than conventional RNA IP for detection in sensitive immunoblot and proteomics analyses. Another advantage provided by immobilizing the RNP is the convenience of repeating the RNase H treatment and collecting additional eluate. In our experience, 2-4 rounds of RNase H treatment were advisable to collect additional eluate.
In this protocol, key technical concerns are maintaining the optimal temperature and ensuring the sterile handling of the reagents. All reagents should be RNase- and protease-free. The integrity of the RNA sample is a potential pitfall to consider if an RNA-protein interaction is not detectable.
This technique requires the efficient lysis of the cells to access the RNPs in a suitable quantity for detection. While an important caveat is incomplete cell lysis, vigorous treatments may increase non-specific interactions with the RNA. Therefore, the experimental conditions should be measured in order to enrich specific protein-RNA interactions. However, the use of high-stringency washes may eliminate important yet transient interactions. Therefore, the washing conditions are another variable to consider in the specific requirements of a particular experiment.
RIP efficiency is a powerful technique to isolate cognate RNA-protein partners, but some transient or weak interactions that are of physiological importance are lost during the binding or washing steps. Cross-linking of the mRNP complex is an option to consider in an analysis. Moreover, physiological growth conditions should be kept in mind while analyzing regulatory RNPs, as the expression level of the cognate RNA or RBP may be subject to physiological fluctuations under certain conditions. Furthermore, the type of downstream analysis will also play a role in selecting the lysis and washing conditions during the immunoprecipitation.
In addition, the successful immunoprecipitation requires that the cell lysate be significantly dilute (at least 1:1). 1x wash buffer (20 mM Tris-HCl, pH 7.3; 3 mM MgCl2; and 150 mM NaCl) is the selected diluent, as its composition does not affect RNP integrity, solution solubility, or antibody-antigen interactions.
For downstream mass spectrometry, samples should be free from salts, including Na+, Cl-, and Tris, as well as some detergents. If necessary, components of buffers-the ionic compounds-may be reduced by dialysis or ionic exchange filtration, and in some cases, volatile buffers are useful in the final steps. In cases when antiserum is used to isolate an epitope-tagged protein expressed by transfection, Western blot validation of the recombinant protein using initial cell lysate should be executed before further analysis. In case an epitope tag is used (i.e., FLAG, MYC, GFP, etc.), the exposure of the tag is crucial to the success of the antibody binding step. Freeze-thaw of samples should be avoided.
The RIP technique has been widely employed to elucidate diverse mechanisms of post-transcriptional gene control10,25. This includes the identification of novel protein-mRNA, protein-microRNA, and protein-protein interactions10,25. Here, we provide a comprehensive protocol for the determination of RNP composition within a cell culture. Our method allows for the targeted and genome-wide assessment of critical protein-RNA interactions, as well as for the capture of rare and/or transient RNP complexes.
The application of this technique has contributed to our characterization of RNAs bound by DHX9/RNA helicase A and helped us to define the critical post-transcriptional regulation of retroviruses and the cellular proto-oncogenes junD6,26 and huR (Fritz and Boris-Lawrie, unpublished data). We expect the application of this technique in future studies to further our understanding of the critical mechanisms for gene control, including those mediated by long non-coding RNP complexes.

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><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&amp;nbsp; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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 ribonucleoprotein complexes (RNPs) formed in proliferating cells, we performed FLAG-MS2 RNP immunoprecipitation in transfected HEK293 cell lysates. The results show efficient precipitation of the FLAG-MS2 protein. Notably, DHX9/RHA is identified within this RNP complex and not within the IgG isotype control, nor within the negative-control HEK293 cell lysate (Figure 2A). The matrix containing the RNPs was incubated with a complementary 30-nt oligonucleotide, and then an RNase H cleavage of the RNA-DNA hybrid was performed. Upon RNase H digestion, the eluates were analyzed by Western blotting. The demonstrated RNase H cleavage of PCEgag RNA at the RNA-DNA hybrid position released the RNP complex specifically bound to the 5&#39; UTR. The DHX9/RHA-associated RNPs were determined to be enriched in the eluents. Importantly, the FLAG-MS2-bound RNPs remained with the antibody-conjugated beads (Figure 2B). The co-immunoprecipitation of the MS2 stem-loop containing retroviral PCEgag RNA in cells and in eluents was validated by RT-PCR and qRTPCR (Figure 2C). The results confirmed a select association between DHX9/RHA and the retroviral 5&#39; UTR, which has been highlighted as a unique RNP important for targeted translation control6.
<img alt="Figure 1" src="/files/ftp_upload/54391/54391fig1.jpg" /> 
Figure 1: RNP isolation and the enrichment of specific RNPs associated with the 5&#39; UTR of PCEgag by RNase H cleavage. Workflow showing the steps to isolate a selected ribonucleoprotein formed de novo in cells, its collection by oligonucleotide-guided RNaseH cleavage, and the downstream analysis of RNA and protein components. Summary of affinity chromatography using a high-affinity interaction between multimers for the MS2 RNA stem-loop and the MS2 coat protein-FLAG fusion protein to capture PCE RNA on FLAG beads. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54391/54391fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54391/54391fig2.jpg" /> 
Figure 2: A PCEgag RNA containing 6 MS2 RNA binding sites was captured by an immobilized FLAG-tagged MS2 coat protein, and specific RNPs associated with the 5&#39; UTR were released by oligonucleotide-directed RNase H cleavage. HEK293 cells were co-transfected with pAR200 (a plasmid containing the PCEgag and 6x MS2 loops in the HIV intron) and a plasmid expressing the FLAG epitope-tagged MS2 protein with a nuclear localization sequence (NLS). Cells were collected and the cytoplasm was isolated at 48 hr post-transfection. The cytoplasmic lysates were subjected to immunoprecipitation with either FLAG antibody or an IgG control. (A)&#160;The IP efficiency was determined by immunoblot using an anti-FLAG antibody. DHX9/RHA, a PCE binding protein, served as a positive control; PCE-containing RNA immunoprecipitated with the MS2 protein. Lanes 1-3: Input proteins used for IP. Lanes 4-5: FLAG IP of FLAG MS2 overexpressed lysate and HEK293 cytoplasmic cell lysate. Lane 6: IgG IP of FLAG MS2 overexpressed lysate. Lanes 7-9: Flow-through of the IPs. (B)&#160;5&#39; UTR-bound RNPs were enriched by RNase H cleavage. Lanes 1-3: Eluates of the 5&#39; UTR-bound protein by RNase H. Lanes 4-6: proteins bound to antibody-conjugated beads. (C)&#160;Reverse transcriptase PCR and real-time quantitative PCR of specific RNA bound to different fractions of RNPs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54391/54391fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

isolation-cognate-rna-protein-complexes-from-cells-using

Nanyang Technological University

Research

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

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

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

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Genetics

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

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 ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

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 ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

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

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

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

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Biology

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

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

Source: Singh, R. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59635">Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins.</a> J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.