This work was funded by Singapore NRF fellowship (NRF-2011NRF-NRFF001-025) as well as CBRG (NMRC/CBRG/0070/2014).

NOTE: The RNA of interest in the context of this study is a fragment of AR 3&#39;UTR.
1. Preparation of Biotin-labeled RNA
<ol>
	<li>To obtain the RNA, amplify T7-AR-oligo by PCR, followed by in vitro transcription using a commercial kit and following manufacturer&#39;s instructions11. 
	NOTE: Primers for PCR are listed in Table 4: T7-AR-F and T7-AR-R.</li>
	<li>For non-specific binding control, amplify a partial sequence of the firefly luciferase (FL) DNA by PCR from psiCHECK-2 plasmid, followed by in vitro transcription. 
	NOTE: Primers for PCR are listed in Table 4: hluc(+)-T7-probe-F and hluc(+)-probe-R. Table 4. Sequences of template and primers for PCR amplification.</li>
	<li>For a 50 &#181;l PCR reaction, mix 50 ng DNA (oligo or plasmid), 4 &#181;l dNTPs (2.5 mM of each individual dNTP), 5 &#181;l 10x reaction buffer, 1 &#181;l 2.5 U/&#181;l DNA polymerase, 2 &#181;l 10 &#181;M forward primer, 2 &#181;l 10 &#181;M reverse primer, and nuclease-free water.
	<ol>
		<li>Run PCR amplification in a thermal cycler using the following program. STEP1: one cycle of 95 &#176;C for 2 min; STEP2: 35 cycles of 95 &#176;C for 30 sec, 55 &#176;C for 30 sec and 72 &#176;C for 30 sec (AR) or 60 sec (FL); STEP3: one cycle of 72 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>Synthesize biotinylated RNAs using an in vitro transcription kit following manufacturer&#39;s instructions11. In this experiment, use PCR products as templates for RNA synthesis. For a 20 &#181;l in vitro transcription reaction, mix 200 ng PCR amplified DNA, 2 &#181;l of each individual NTP, 1 &#181;l 10mM biotin-CTP, 2 &#181;l 10x reaction buffer, and 2 &#181;l T7 RNA polymerase. Incubate the mixture at 37 &#176;C for 4 hr.</li>
	<li>Following in vitro transcription, treat reaction mixture with 1 &#181;l 2 U/&#181;l DNase at 37 &#176;C for 15 min to degrade template DNA. Finally, dilute the biotinylated RNA to a total volume of 60 &#181;l for subsequent purification.</li>
	<li>Purify biotinylated RNAs to remove salts and unincorporated nucleotides by using purification columns following manufacturer&#39;s instructions12. Measure RNA concentration and store at -80 &#176;C for subsequent pull-down assays.</li>
	<li>To ensure proper secondary structure for RNA of interest in RNA-protein interaction, heat 50 &#181;l biotinylated RNA (50 pM) at 90 &#176;C for 2 min, chill on ice for 2 min, then supply with 50 &#181;l 2x RNA structure buffer (Table 3), and allow for the RNA folding at RT for 30 min13,14.</li>
</ol>
2. Preparation of RNA-conjugated Beads
NOTE: Use a magnetic stand for separation of the magnetic beads and supernatant.
<ol>
	<li>Resuspend the streptavidin-coated magnetic beads in the original vial by brief vortexing following manufacturer&#39;s instructions. Transfer 150 &#181;l resuspended beads to a 1.5 ml tube. Place the tube on magnet for 1 min. Discard supernatant. Keep bead pellet. 
	NOTE: In RNA pull-down assays, when aliquoting resuspended beads from original vial, use 50 &#181;l beads for an assay followed by western blotting, and 200 &#181;l beads for an assay followed by MS.</li>
	<li>Wash beads once by re-suspending bead pellet with 1 ml of the manufacturer&#39;s recommended 1x W&#38;B (binding and washing) buffer (Table 3), followed by rotating the tube for 5 min at room temperature in a rotator. Discard supernatant. Keep bead pellet.</li>
	<li>To inactivate RNase on beads, wash beads twice, each time by re-suspending bead pellet with 400 &#181;l buffer A. Buffer A is an alkaline solution containing 0.1 M NaOH and 0.05 M NaCl (Table 3). Rotate the tube for 2 min at room temperature in a rotator. Discard supernatant. Keep bead pellet.</li>
	<li>To remove NaOH from beads, wash beads twice, each time by re-suspending bead pellet with 400 &#181;l buffer B (Table 3), followed by rotating the tube for 2 min at room temperature in a rotator. Discard supernatant. Keep bead pellet.</li>
	<li>Resuspend bead pellet with 300 &#181;l 2x W&#38;B buffer, split beads equally and transfer into three 1.5 ml tubes (100 &#181;l beads per tube). Supply the bead-containing tubes with 100 &#181;l RNase free water, 100 &#181;l biotinylated FL RNA and 100 &#181;l biotinylated AR 3&#39;UTR RNA, respectively.</li>
	<li>Incubate each bead mixture for 1 hr at room temperature with rotation. Place tubes on magnet for 1 min. Discard all supernatant. Keep bead pellets.</li>
	<li>Wash beads twice, each time by re-suspending each bead pellet with 400 &#181;l 1x W&#38;B buffer, followed by rotating tubes for 5 min at room temperature in a rotator.</li>
	<li>Place tubes on magnet for 1 min. Discard all supernatant. Resuspend each bead pellet with 50 &#181;l nuclear isolation buffer (Table 3).</li>
</ol>
3. Preadipocyte Isolation and Adipogenesis Induction
<ol>
	<li>As described in previous publications5, dissect preadipocytes from the interscapular brown fat pad (C57BL6 wild type mice, 3 weeks old).</li>
	<li>Grow and in vitro differentiate the preadipocytes5. Cells are ready for downstream application 4-5 days after initiation of differentiation.</li>
</ol>
4. Preparation of Cellular Lysate from Primary Adipocytes
NOTE: Ensure all procedures of cell lysate preparation are made on ice, and all buffers are chilled on ice. Pre-chill dounce glass homogenizers on ice.
<ol>
	<li>Grow and differentiate preadipocytes in 15 cm dishes5 (see section 3.2). Each dish provides adequate cells for one RNA pull-down assay. On day 4 or day 5 of differentiation, discard cell culture medium, and wash cells once with 1x PBS.</li>
	<li>To harvest cells, add 10-15 ml 1x PBS to cells, scrape to collect cells in a 50 ml tube, and pellet cells by centrifugation at 200 x g for 5 min at RT. Discard supernatant by using 1 ml pipette.</li>
	<li>Resuspend cell pellet in 2 ml hypotonic buffer (Table 3), and incubate cells on ice for 15 min. Transfer cells to a 15 ml dounce glass homogenizer, and shear cells mechanically on ice with 20 strokes.</li>
	<li>Pellet cell nuclei by centrifugation at 3,300 x g for 15 min at 4 &#176;C. Keep nuclei pellet on ice for further use (see section 4.6). Transfer supernatant to 1.5 ml tubes, add 3M KCl to supernatant to obtain a final concentration of 150 mM, and spin at 20,000 x g for 15 min at 4 &#176;C.</li>
	<li>Following centrifugation, carefully remove lipidlayeron the top by using 1 ml pipette, and collect supernatant as the cytoplasmic cellular lysate.</li>
	<li>Resuspend nuclei pellet (see section 4.4) in 1 ml nuclear isolation buffer. Transfer nuclei to a 15 ml dounce glass homogenizer using 1 ml pipette, and shear nuclei mechanically on ice with 100 strokes. Pellet nuclear debris by centrifugation at 20,000 x g for 15 min at 4 &#176;C, and collect supernatant as the nuclear cellular lysate.</li>
	<li>Either combine the nuclear and cytoplasmic cellular lysates, or use each fraction separately, for downstream pull-down application. In this demo experiment, these two fractions of cell lysate were combined at a volume ratio of 1:1.</li>
	<li>Add RNase inhibitor to this unfractionated cell lysate to obtain a final concentration of 0.5 U/&#181;l. Set aside 100 &#181;l of cell lysate as input control for western blotting15.</li>
</ol>
5. Binding and Elution of RBP
<ol>
	<li>Split cell lysate into three equal aliquots (1ml cell lysate in a 1.5 ml tube), and incubate with blank, FL RNA-conjugated and AR 3&#39;UTR RNA-conjugated beads (see section 2.8), respectively, for 3 hr at 4 &#176;C with rotation.</li>
	<li>Place tubes on magnet for 1 min. Collect supernatant using a 1 ml pipette, followed by RNA isolation from supernatant to confirm RNA integrality. Isolate RNA by using an acid guanidinium thiocyanate-phenol solution following manufacturer&#39;s instructions (Table 2). Assess the RNA intactness by analyzing 28S and 18S subunits of ribosomal RNA. Keep bead pellets on ice.</li>
	<li>Wash the magnetic beads six times, each time by resuspending each bead pellet with 1 ml nuclear isolation buffer containing 40 U RNase inhibitor and 0.25% IGEPAL, followed by rotating tubes for 2 min at room temperature in a rotator. Use a magnetic stand for separation of the magnetic beads and supernatant. Discard all supernatant. Keep bead pellets on ice.</li>
	<li>Use following steps to elute RNA-protein complexes from beads for western blotting. First, resuspend each bead pellet in 75 &#181;l nuclear isolation buffer; then, add 25 &#181;l 4x western blot loading buffer (containing SDS) to obtain a total volume of 100 &#181;l. Finally, boil beads in this 100 &#181;l solution at 95 &#176;C for 5 min. 
	NOTE: Use following steps to elute RNA-protein complexes from beads for MS. STEP1: resuspend each bead pellet in 100 &#181;l elution buffer (Table 3); STEP2: incubate bead samples for 2-3 hr at room temperature with rotation; STEP3: centrifuge and collect protein-containing supernatant; STEP4: concentrate protein solutions to 20-30 &#181;l before submission for MS.</li>
	<li>Centrifuge each 100 &#181;l of bead sample at 12,000 x g for 30 sec at 4 &#176;C, and collect supernatant. Aliquot 15 &#181;l supernatant for western blot analysis (see section 6.1 and 6.2). Probe the resulting membrane with the diluted HuR mouse monoclonal antibody (1:1,000 dilution), overnight.</li>
</ol>
6. Western Blotting for Verification of RBP
<ol>
	<li>Separate RBPs by SDS-PAGE using standard techniques.</li>
	<li>Conduct western blotting using standard techniques by probing for the RBPs associated with the RNA of interest. 
	NOTE: In this demo experiment, HuR mouse monoclonal antibody was used for the immunodetection of HuR protein.</li>
	<li>Incubate the resulting membrane with the diluted HuR antibody (1:1,000 dilution) in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween-20 at 4 &#176;C with gentle shaking, overnight. Wash membrane three times with 1x TBST. Incubate membrane with the secondary antibody (goat anti-mouse IgG-HRP) at room temperature for 1 hr. Wash membrane. Develop and record signal.</li>
</ol>

<ol>
	<li>Huot, M. &. #. 2. 0. 1. ;., 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=The+Sam68+STAR+RNA-binding+protein+regulates+mTOR+alternative+splicing+during+adipogenesis.">The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.</a> Mol Cell. 46 (2), 187-199 (2012).</li><li>Sun, 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=Long+noncoding+RNAs+regulate+adipogenesis.">Long noncoding RNAs regulate adipogenesis.</a> Proc Natl Acad Sci U S A. 110 (9), 3387-3392 (2013).</li><li>Engreitz, J. 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=The+Xist+lncRNA+exploits+three-dimensional+genome+architecture+to+spread+across+the+X-chromosome.">The Xist lncRNA exploits three-dimensional genome architecture to spread across the X-chromosome.</a> Science. 341 (6147), 1237973 (2013).</li><li>Zhao, X. Y., Li, S., Wang, G. X., Yu, Q., Lin, 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=A+long+noncoding+RNA+transcriptional+regulatory+circuit+drives+thermogenic+adipocyte+differentiation.">A long noncoding RNA transcriptional regulatory circuit drives thermogenic adipocyte differentiation.</a> Mol Cell. 55, 372-382 (2014).</li><li>Alvarez-Dominguez, J. 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=De+novo+reconstruction+of+adipose+tissue+transcriptomes+reveals+long+non-coding+RNA+regulators+of+brown+adipocyte+development.">De novo reconstruction of adipose tissue transcriptomes reveals long non-coding RNA regulators of brown adipocyte development.</a> Cell Metab. 21, 764-776 (2015).</li><li>Adams, D. J., Beveridge, D. J., van der Weyden, L., Mangs, H., Leedman, P. J., Morris, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HADHB,+HuR,+and+CP1+bind+to+the+distal+3-untranslated+region+of+human+renin+mRNA+and+differentially+modulate+renin+expression.">HADHB, HuR, and CP1 bind to the distal 3-untranslated region of human renin mRNA and differentially modulate renin expression.</a> J Biol Chem. 278 (45), 44894-44903 (2003).</li><li>Yeap, B. B., 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=Novel+binding+of+HuR+and+poly(C)-binding+protein+to+a+conserved+UC-rich+motif+within+the+3&#180;+untranslated+region+of+the+androgen+receptor+messenger+RNA.">Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3&#180; untranslated region of the androgen receptor messenger RNA.</a> J Biol Chem. 277, 27183-27192 (2002).</li><li>K&#246;nig, J., Zarnack, K., Luscombe, N. M., Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-RNA+interactions:+new+genomic+technologies+and+perspectives.">Protein-RNA interactions: new genomic technologies and perspectives.</a> Nat Rev Genet. 13, 77-83 (2012).</li><li>Ferr&#232;, F., Colantoni, A., Helmer-Citterich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+protein-lncRNA+interaction.">Revealing protein-lncRNA interaction.</a> Briefings in Bioinformatics. , 1-11 (2015).</li><li>McHugh, C. A., Russell, P., Guttman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+comprehensive+experimental+identification+of+RNA-protein+interactions.">Methods for comprehensive experimental identification of RNA-protein interactions.</a> Genome Biol. 15 (1), 203 (2014).</li><li>Lee, Y. S., 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=AU-rich+element-binding+protein+negatively+regulates+CCAAT+enhancer+binding+protein+mRNA+stability+during+long+term+synaptic+plasticity+in+Aplysia.">AU-rich element-binding protein negatively regulates CCAAT enhancer binding protein mRNA stability during long term synaptic plasticity in Aplysia.</a> Proc Natl Acad Sci U S A. 109 (38), 15520-15525 (2012).</li><li>Hermann, M., Cermak, T., Voytas, D. F., Pelczar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genome+engineering+using+designer+nucleases.">Mouse genome engineering using designer nucleases.</a> J Vis Exp. (86), (2014).</li><li>Tsai, M. 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=Long+Noncoding+RNA+as+Modular+Scaffold+of+Histone+Modification+Complexes.">Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes.</a> Science. 329 (5992), 689-693 (2010).</li><li>Wan, Y., 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=Landscape+and+variation+of+RNA+secondary+structure+across+the+human+transcriptome.">Landscape and variation of RNA secondary structure across the human transcriptome.</a> Nature. 505, 706-709 (2014).</li><li>Li, H., 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=Identification+of+mRNA+binding+proteins+that+regulate+the+stability+of+LDL+receptor+mRNA+through+AU+rich+elements.">Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU rich elements.</a> J Lipid Res. 50 (5), 820-831 (2009).</li><li>McHugh, C. A., 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=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li><li>Chu, 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=Systematic+Discovery+of+Xist+RNA+Binding+Proteins.">Systematic Discovery of Xist RNA Binding Proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>Chu, C., Spitale, R. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+to+probe+functions+and+mechanisms+of+long+noncoding+RNAs.">Technologies to probe functions and mechanisms of long noncoding RNAs.</a> Nat Struct Mol Biol. 22 (1), 29-35 (2015).</li><li>Zhao, J., 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+identification+of+Polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of Polycomb-associated RNAs by RIP-seq.</a> Mol Cell. 40 (6), 939-953 (2010).</li></ol>

No conflicts of interest declared.

lncRNAs and RBPs have vital roles in health and disease, however the molecular mechanisms of these molecules are poorly understood. Identification of proteins that interact with lncRNA molecules is a key step towards elucidating the regulatory mechanisms. Enrichment of RBPs by the RNA pull-down assay system is based on in-solution capture of interacting complex so that proteins from a cell lysate can be selectively extracted using biotinylated RNA baits bound to streptavidin magnetic beads. Several other methods such as ...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RBPs are proteins that bind to the double or single stranded RNA in cells and participate in forming RNA-protein complexes. RBPs may bind a variety of RNA species, including mRNA and long noncoding RNAs (lncRNAs), and exert their influence at post-transcriptional levels. Both RBPs and lncRNAs are emerging as novel regulators in adipose development and function1,2,3. To understand the mechanism of RBP- and lncRNA-mediated regulation of cellular pathways, it is often necessary to detect the interaction between a specific RNA molecule and one or several RBPs, and, sometimes, to identify the full spectrum of protein partners of a RNA transcript. However, the experiment can be challenging due to the high content of lipids in adipocytes. The RNA pull-down protocol described here can be employed to retrieve the protein partners of a specific RNA from the adipocyte lysates of primary cultures4,5.
Rationale for this protocol is summarized as follows. An RNA bait is in vitro transcribed from an DNA template, biotin-labeled and conjugated to streptavidin-coated magnetic beads4. The RNA bait is incubated with cellular lysates to allow for the formation of RNA-protein complexes, which are subsequently pulled down on a magnetic stand. More specifically, the RNA pull-down protocol described below use an RNA fragment from AR 3&#39;UTR as the bait to retrieve HuR protein, a universally expressed RBP bound to 3&#39;UTR of mRNAs6,7, from theadipocytelysate of primary culture. To test the binding specificity of this assay, a non-relevant RNAas well as blank streptavidin beads is included as control. This protocol is compatible with western blotting or mass spectrometry (MS) to confirm the capture of a specific RBP or to identify the full repertory of captured RBPs, respectively8.
Several techniques are available to study RNA-protein interactions. To reveal RNAs bound by a given RBP, RIP (RNA immunoprecipitation) and CLIP (UV crosslinking and immunoprecipitation) can be applied. In contrast, to identify protein partners of a given RNA, RNA pull-down, ChIRP (chromatin isolation by RNA purification), CHART (capture hybridization analysis of RNA targets) and RAP (RNA antisense purification) can be applied. In comparison with the later ones, RNA pull-down technique takes less effort to set up. It can be employed to capture proteins in vitro and in vivo. The in vivo approach of RNA pull-down, which is technically more challenging than its in vitro counterpart, preserves RNA-protein interactions by crosslinking in cells, captures aptamer-tagged RNAs of interest from cells, and subsequently detects bound RBPs. RNA pull-down can be used to enrich low abundant RBPs, and to isolate and identify the RNA-protein complexes that have diverse functional roles in controlling cellular regulation9,10.

<table><tbody><tr><td>&lt;strong&gt;Major materials used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEGAscript kit</td><td>&amp;nbsp;Ambion</td><td></td><td></td></tr><tr><td>Biotin-14-CTP&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>NucAway spin columns&amp;nbsp;</td><td>Ambion</td><td></td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>HuR (3A2) mouse monoclonal antibody (sc-5261)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Goat anti-mouse IgG-HRP(sc-2005)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Hypure Molecular Biology Grade Water (nuclease-free)&amp;nbsp;</td><td>HyClone</td><td></td><td></td></tr><tr><td>Phosphate Buffer Saline (1&amp;times;PBS)</td><td>&amp;nbsp;HyClone</td><td></td><td></td></tr><tr><td>RNase inhibitor&amp;nbsp;</td><td>Bioline</td><td></td><td></td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma</td><td></td><td></td></tr><tr><td>Pfu Turbo DNA polymerase&amp;nbsp;</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>TRIsure</td><td>&amp;nbsp;Bioline</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Major equipment used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sorvall Legend Micro 21R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Sorvall Legend X1R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bio-Rad T100 thermal cycler&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Nanodrop 2000&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>BOECO rotator Multi Bio RS-24&amp;nbsp;</td><td>BOECO,Germany</td><td></td><td></td></tr><tr><td>Protein gel electrophoresis and blotting apparatus&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>DynaMag-2 magnet&amp;nbsp;</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

In this demo experiment, an AR RNA fragment was used as the bait to capture its binding protein HuR. Both a FL RNA bait that is non-relevant to HuR protein, and an aliquot of unconjugated blank streptavidin beads served as negative controls. RNA-protein interactions can occur either in nucleus or cytoplasm, and this pull-down protocol can be applied to either total or fractionated (nuclear or cytoplasmic) cell lysates. Analysis of proteins by western blotting shows that the sample of 15 &...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

23.5K Views.  Duke-NUS Medical School. An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Video: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

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

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Biochemistry

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

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 ...

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Cancer Research

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

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 ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Summary

Explore More Videos

Chapters in this video

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

mRNA Interactome Capture from Plant Protoplasts

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

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

An Assay for Quantifying Protein-RNA Binding in Bacteria

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

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

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function