We thank Prof. Jeff Gerst and Boris Slobodin for their helpful advice in setting up the RaPID protocol and providing the necessary plasmids. We also thank Dr. Avigail Atir-Lande for her help in establishing this protocol and Dr. Tamar Ziv from the Smoler Proteomics Center for her help with the LC-MS/MS analysis. We thank Prof. T.G. Kinzy (Rutgers) for the YEF3 antibody. This work was supported by grant 2011013 from the Binational Science Foundation.

Note: Insert a sequence consisting of 12 MS2-binding sites (MS2 loops; MS2L) into the desired genomic locus, usually between the open reading frame (ORF) and the 3&#39; UTR. A detailed protocol for this integration is provided elsewhere22. Verify proper insertion and expression by PCR, northern analysis or RT-PCR20,23. It is important to verify that the integration did not intervene with the synthesis of the 3&#39;UTR. In addition, a plasmid-expressing MS2-CP fused to SBP under the expression of an inducible promoter (methionine depletion) should also be introduced into the cells24. An identical strain, excluding the introduced MS12 loops, should be used as a control for the detection of non-specific signals.
1. RaPID Purification
<ol>
	<li>Grow 500 ml of yeast cells (use 250-1,000 ml of yeast cells depending on the expression level of the gene of interest) expressing MS2-tagged mRNA20 and MS2-CP-GFP-SBP fusion protein24 to OD600 0.8 - 1.0 at 30 oC in the appropriate growth medium (e.g., Synthetic Dextrose (SD) selective medium).</li>
	<li>Centrifuge cells at 3,000 x g for 4 min at RT&#160;and discard the supernatant.</li>
	<li>Wash cells in 1x phosphate buffered saline (PBS), centrifuge as before (step 1.2), resuspend in an equal volume (step 1.1) of SD without methionine, and incubate for 45 - 60 min at 30 oC to induce expression of the MS2-CP-GFP-SBP fusion protein. 
	Note: Longer induction time may cause aggregation of GFP.</li>
	<li>Set aside 10 ml of cells and extract RNA from this sample using the simple &#34;hot phenol&#34; method25. Caution: Phenol is highly toxic and should be handled according to its safety instructions. 
	Note: This RNA is a good reference for the quality of isolated RNA (Figure 1A).</li>
	<li>For 500 ml cells, add 1.35 ml 37% formaldehyde to a final concentration of 0.1% to crosslink the RNA-protein complexes. Continue incubation at 30 oC for 10 min. Caution: Formaldehyde is highly toxic and should be handled according to its safety instructions.</li>
	<li>Add 26.5 ml of 2.5 M glycine to a final concentration of 0.125 M and incubate for 3 min to stop crosslinking. From this step on, put everything on ice to minimize RNA degradation.</li>
	<li>Centrifuge cells at 3,000 x g for 4 min at 4oC and wash with 45 ml cold 1x PBS.</li>
	<li>Resuspend cells in 1 ml RaPID Lysis buffer per 100 ml of initial cell culture.</li>
	<li>Split into aliquots of 500 &#181;l in screw-capped microfuge tubes and add ~ 400 mg of chilled glass beads to each aliquot (roughly a full 0.2 ml PCR tube).</li>
	<li>Lyse cells in a bead beater for 3 min. Immediately put the lysate on ice. 
	Note: Cell lysis releases cellular RNases, which are the major cause of RNA degradation. RaPID Lysis buffer contains a high concentration of RNase inhibitors, such as the nonspecific RNase inhibitor heparin and specific inhibitors. Working quickly and keeping everything cold is also recommended.</li>
	<li>Transfer the lysate to new tubes. Pierce a small hole in the bottom of each microfuge tube with a hot needle (0.8 mm x 40 mm) and place the microfuge tubes on top of the 15 ml tubes. Use the head of a 5 ml syringe as an adaptor between the microfuge tubes and the 15 ml tubes. Centrifuge the tubes assembly at 3,000 x g for 1 min at 4 oC.</li>
	<li>Transfer the flow-through from the 15 ml tube to a new 1.5 ml tube and centrifuge at 10,000 x g for 10 min at 4 oC to clear cell debris.</li>
	<li>Pool supernatants from all 1.5 ml tubes into a single 15 ml tube, and set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively. 
	Note: These will serve as the &#34;Input&#34; samples for subsequent analysis (Figure 1).</li>
	<li>Add 300 &#181;g of avidin per 500 ml of initial yeast culture and incubate for 30 min (this can be reduced to 10 min) at 4 oC under constant rolling (10 rpm). 
	Note: Avidin blocks biotin and biotinylated proteins from binding to the streptavidin beads.</li>
	<li>Pre-wash the streptavidin beads before incubation with the cell lysate.
	<ol>
		<li>Transfer about 300 &#181;l of the streptavidin beads slurry to a 1.5 ml microcentrifuge tube and remove the upper supernatant. This will result in 250 &#181;l of beads. Wash the beads twice with 1 ml of RaPID lysis buffer. Centrifuge the streptavidin beads at 660 x g for 2 min at 4oC between each step.</li>
		<li>Block the beads by incubating for 1 hr with 0.5 ml of RaPID lysis buffer, 0.5 ml of 10 mg/ml BSA and 10 &#181;l of 10 mg/ml yeast tRNA. Beads can be left O/N in blocking solution at 4oC with rotation if necessary.</li>
		<li>Wash twice with 1 ml of RaPID Lysis buffer. 
		Note: Magnetic beads can also be used to reduce time and background. These, however, have lower capacity than sepharose beads.</li>
	</ol>
	</li>
	<li>Add 250 &#181;l of the pre-washed streptavidin beads to the avidin-containing lysate (from step 1.14) and incubate 1 hr at 4 &#176;C with constant rotation (10 rpm). 
	Note: This incubation time is a compromise between providing sufficient binding time and minimizing chances for RNA degradation.</li>
	<li>Centrifuge at 660 x g for 2 min at 4 oC to clear the streptavidin beads and transfer the supernatant to a new 15 ml tube. Set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively. 
	Note: These will be the &#34;Unbound&#34; samples.</li>
	<li>Wash the beads with 1 ml of RaPID Lysis buffer, shake gently, transfer to a new 1.5-ml microcentrifuge tube, and centrifuge as in step 1.17. Repeat this step three times.</li>
	<li>Wash the beads with 1 ml of RaPID Wash buffer, and this time rotate (10 rpm) for 5 min at 4 &#176;C. Repeat this step twice.</li>
	<li>Wash the beads with 1 ml of 1x PBS and centrifuge as above.</li>
	<li>Wash the beads again with 120 &#181;l of 1x PBS. Centrifuge as above, and take the entire supernatant for RNA and protein extraction as the &#34;Wash&#34; sample. 
	Note: This last wash sample will indicate the stringency of the washes and should be of the same volume as the Elution sample. This will simplify processing and loading in subsequent assays.</li>
	<li>To elute RNA and RNA-associated proteins from the column, add 120 &#181;l of 6 mM biotin in 1x PBS and incubate for 45 min at 4 &#176;C with constant rotation (10 rpm).</li>
	<li>Centrifuge the beads as above and transfer the eluted material to a new 1.5 ml tube. Spin the eluted sample again and transfer the upper phase to a new 1.5 ml tube to ensure no carryover of beads.</li>
	<li>Take samples for RNA or protein extraction. 
	Note: The volume taken should depend on the following assay and expression level of the RNA or protein of interest. As a guideline, for northern analysis26, take 80% of the sample, for Western blot23,27 or RT-PCR28, take 20%, and for mass spectrometry29 to discover new proteins, take the entire sample.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Add an equal volume of 2x Crosslinking Reversal Buffer and incubate for 45 min at 65 &#176;C. Continue directly for RNA extraction. 
	Note: The Crosslinking Reversal Buffer contains ethylenediaminetetraacetic acid (EDTA), which significantly improves RNA extraction20.</li>
	<li>Use the standard phenol:chloroform method26 to extract RNA from the &#34;input&#34; and &#34;unbound&#34; samples (steps 1.13 and 1.17). 
	Note: These samples contain a large amount of the RNase inhibitor heparin, which can inhibit subsequent Reverse Transcriptase-(RT)utilizing assays (e.g., RT-PCR), therefore, LiCl precipitation30 is advised to remove it. The &#34;wash&#34; and &#34;elution&#34; samples (steps 1.21 and 1.24) contain low amounts of RNA and are precipitated through the following steps.</li>
	<li>Add an equal volume of 8M Guanidinium-HCl (GuHCl) and two volumes of 100% ethanol to the &#34;wash&#34; and &#34;elution&#34; samples. Incubate at -80 &#176;C for at least 2 hr. 
	Note: Guanidinium will efficiently denature proteins and thereby inhibit RNases and proteases in the sample.</li>
	<li>Centrifuge at 20,000 x g, 4 &#176;C for 30 min and discard the supernatant. Wash the pellet with cold 80% ethanol and centrifuge again at 20,000 x g, 4 &#176;C for 15 min.</li>
	<li>Dissolve the RNA pellet in 400 &#181;l of RNase free water and precipitate again to remove any leftover GuHCl or other contaminants. Add 40 &#181;l of 3 M sodium acetate, pH 5.2 and 800 &#181;l of 100% ethanol, incubate at -20 &#176;C for at least 1 hr.</li>
	<li>Centrifuge and wash as in step 2.4 and remove all ethanol with a 10 &#181;l tip. Resuspend the RNA pellet in 10 &#181;l RNase free water. Take extra care as the RNA pellet is usually not visible. 
	Note: Samples can be used for northern analysis as described in23 (Figure 1).</li>
</ol>
3. Protein Preparation for Western Blot or Mass Spectrometry Analysis
<ol>
	<li>Add 1/3 volume of 4x Laemmli Sample Buffer (LSB) to protein samples and incubate 45 min at 65 &#176;C to reverse the crosslinking of RNA and proteins. 
	Note: Samples can be stored at -20 &#176;C.</li>
	<li>Run proteins on a SDS polyacrylamide gel (SDS-PAGE)31. Adjust the percentage of the gel according to the size of proteins to be analyzed. 
	Note: 10% acrylamide gel gives a wide range of separation and is good as a first approximation.</li>
	<li>Transfer proteins to the membrane as in the standard immunoblot analysis27 for control of isolation efficiency (Figure 1C). Stain with either Coomassie Blue R250 or silver stain before mass spectrometry analysis. 
	Note: Silver stain is the preferred choice because it is more sensitive and allows better detection (Figure 1B). Staining can be done with either manually prepared solutions or commercial kits. The incubation time of the gel in the final dye solution should be determined experimentally. Long incubation may reveal low abundant proteins, but can cause highly expressed proteins such as MS2-CP-SBP-GFP to be over stained and may mask surrounding proteins in their vicinity. Additionally, a high background might occur. Follow the reaction carefully, and add Stop Solution at the appropriate time.</li>
	<li>Cut out bands from the gel and transfer them to 1.5 ml tubes. Store samples at -20 &#176;C, or send for protein extraction and trypsin digestion followed by LC-MS/MS analysis32.</li>
	<li>As an alternative proteomic approach to exclude the gel separation step, digest eluted material from step 1.24 in solution using trypsin and subject to LC-MS/MS analysis33. 
	Note: Omitting the SDS-PAGE separation simplifies the protocol and increases yield. However, samples might contain traces of the detergent NP-40, which inhibits mass spectrometry analysis. To overcome this problem, perform the following steps:
	<ol>
		<li>Run protein on SDS-PAGE31 for 10 - 15 min.</li>
		<li>Cut out the entire portion of the lane (i.e., where the proteins are located). 
		Note: The protein sample might include a significant amount of the MS2-CP-SBP protein that may obscure signals of low abundance proteins.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Xenopus+Vg1+mRNA+by+Vera+protein+and+the+endoplasmic+reticulum.">Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum.</a> Science. 276, 1128-1131 (1997).</li><li>Bertrand, E., 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=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol Cell. 2, 437-445 (1998).</li><li>Aizer, 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=Quantifying+mRNA+targeting+to+P-bodies+in+living+human+cells+reveals+their+dual+role+in+mRNA+decay+and+storage.">Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage.</a> J Cell Sci. 127, 4443-4456 (2014).</li><li>Gadir, N., Haim-Vilmovsky, L., Kraut-Cohen, J., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+mRNAs+coding+for+mitochondrial+proteins+in+the+yeast+Saccharomyces+cerevisiae.">Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.</a> RNA. 17, 1551-1565 (2011).</li><li>Lopez de Heredia, ., M, R. P., Jansen, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+integrity+as+a+quality+indicator+during+the+first+steps+of+RNP+purifications+:+a+comparison+of+yeast+lysis+methods.">RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.</a> BMC Biochem. 5, 14 (2004).</li><li>Lee, J. S., Kallehauge, T. B., Pedersen, L. E., Kildegaard, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+in+CHO+cells+mediated+by+CRISPR/Cas9+and+homology-directed+DNA+repair+pathway.">Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway.</a> Sci Rep. 5, 8572 (2015).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome Res. 24, 142-153 (2014).</li><li>Oda, Y., Huang, K., Cross, F. R., Cowburn, D., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantitation+of+protein+expression+and+site-specific+phosphorylation.">Accurate quantitation of protein expression and site-specific phosphorylation.</a> Proc Natl Acad Sci USA. 96, 6591-6596 (1999).</li><li>de Godoy, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILAC+yeast:+from+labeling+to+comprehensive+proteome+quantification.">SILAC yeast: from labeling to comprehensive proteome quantification.</a> Methods Mol Biol. 1156, 81-109 (2014).</li></ol>

The authors declare no competing financial interests.

Various methods use the isolation of specific mRNAs to identify their associated proteins11,34 35. These methods apply in vitro and in vivo strategies to probe RNA-protein interactions. In vitro methods incubate exogenously transcribed RNA with cell lysate to capture RBPs and isolate RNP complexes36,37. An effective approach of this type was presented recently, which enabled the identification of novel proteins that bind a regulatory RNA motif18. A dr...

RNA-binding proteins (RBPs) represent about 10% of S. cerevisiae proteins1,2 and about 15% of mammalian proteins3-5. They are implicated in many cellular processes such as mRNA post-transcriptional processing and regulation, translation, ribosome biogenesis, tRNA aminoacylation and modification, chromatin remodeling, and more. An important subgroup of RBPs is the mRNA-binding proteins (mRNPs)6,7. In the course of mRNA maturation, different RBPs bind the transcript and mediate its nuclear processing, export out of the nucleus, cellular localization, translation and degradation6-8. Thus, the distinct set of RBPs bound to a particular transcript at any time point determines its processing and ultimately its fate.
The identification of RBPs associated with an mRNA could significantly improve our understanding of processes underlying their post-transcriptional regulation. Diverse genetic, microscopic, biochemical and bioinformatics methods have been used to identify proteins involved in mRNA regulation (reviewed in9-11). However, only a few of these methods enable the identification of proteins associated with a particular target mRNA. Of note is the Yeast Three Hybrid system (Y3H), which utilizes the mRNA of interest as bait to screen an expression library in yeast cells. Positive clones are usually observed through a growth selection or reporter expression12-14. The key advantage of this method is the large number of proteins that can be scanned in a cellular environment and the ability to measure the strength of the RNA-protein interaction. Drawbacks include the relatively large number of false positive results due to non-specific binding, and the high potential for false negative results due, in part, to misfolding of the fusion protein prey or the bait RNA.
An alternative to the genetic approach is affinity purification of RNA with its associated proteins. Poly A-containing mRNAs can be isolated through the use of oligo dT columns, and their associated proteins are detected by mass spectrometry. The RNA-protein interaction is conserved in its cellular context by crosslinking, which makes short-range covalent bonds. The use of the oligo dT column yields a global view of the entire proteome that is associated with any poly A-containing mRNA3,5,15. However, this does not provide a list of proteins that are associated with a particular mRNA. Very few methods are available to accomplish such an identification. The PAIR method entails the transfection of nucleic acid with complementarity to the target mRNA16,17. The nucleic acid is also attached to a peptide, which allows crosslinking to RBPs in close vicinity to the interaction site. After crosslinking, the RBP-peptide-nucleic acid can be isolated and subjected to proteomics analysis. Recently, an aptamer-based methodology was successfully applied to extracts from mammalian cell lines18. An RNA aptamer with improved affinity to streptavidin was developed and fused to a sequence of interest (AU-rich element (ARE) in this case). The aptamer-ARE RNA was attached to streptavidin beads and mixed with cell lysate. Proteins that associated with the ARE sequence were purified and identified by mass spectrometry (MS). Although this method detected associations that occur outside the cellular settings (i.e., in vitro), it is likely to be modified in the future so as to introduce the aptamers into the genome and thus enable the isolation of proteins associated with the mRNA while in the cellular milieu (i.e., in vivo). In yeast, where genetic manipulations are well established, the RaPID method (developed in Prof. Jeff Gerst&#39;s lab) provides a view of in vivo associations19. RaPID combines the specific and strong binding of the MS2 coat protein (MS2-CP) to the MS2 RNA sequence, and of the streptavidin-binding domain (SBP) to streptavidin conjugated beads. This enables efficient purification of MS2-tagged mRNAs through streptavidin beads. Moreover, expression of 12 copies of MS2 loops allows up to six MS2-CPs to bind simultaneously to the RNA and increase the efficiency of its isolation. This protocol was therefore suggested to enable the identification of novel mRNA-associated proteins once the eluted samples are subjected to proteomics analysis by mass spectrometry.
We recently utilized RaPID to identify novel proteins associated with the yeast PMP1 mRNA20. PMP1 mRNA was previously shown to be associated with the ER membrane and its 3&#39; untranslated region (UTR) was found to be a major determinant in this association21. Thus, RBPs that bind PMP1 3&#39; UTR are likely to play an important role in its localization. RaPID followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) resulted in the identification of many new proteins that interact with PMP120. Herein, we provide a detailed protocol of the RaPID methodology, important controls that need to be done, and technical tips that may improve yield and specificity.

<table><tbody><tr><td>Tris</td><td>sigma</td><td>T1503</td><td></td></tr><tr><td>SDS</td><td>bio-lab</td><td>1981232300</td><td></td></tr><tr><td>DTT</td><td>sigma</td><td>D9779</td><td></td></tr><tr><td>Acidic Phenol (pH 4.3)</td><td>sigma</td><td>P4682</td><td></td></tr><tr><td>Acidic Phenol: Chloroform (5:1, pH 4.3)</td><td>sigma</td><td>P1944</td><td></td></tr><tr><td>Chloroform</td><td>bio-lab</td><td>3080521</td><td></td></tr><tr><td>Formaldehyde</td><td>Frutarom</td><td>5551820</td><td></td></tr><tr><td>Glycine</td><td>sigma</td><td>G7126</td><td></td></tr><tr><td>NP-40</td><td>Calbiochem</td><td>492016</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Phenylmethylsulfonyl Flouride (PMSF)</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T9003</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>DNase I</td><td>Promega</td><td>M610A</td><td></td></tr><tr><td>Ribonuclease&amp;nbsp; Inhibitor</td><td>Takara</td><td>2313A</td><td></td></tr><tr><td>Glass Beads</td><td>Sartorius</td><td>BBI-8541701</td><td>0.4-0.6mm diameter&amp;nbsp;</td></tr><tr><td>Mini BeadBeater</td><td>BioSpec</td><td>Mini BeadBeater 16</td><td></td></tr><tr><td>Guanidinium</td><td>Sigma</td><td>G4505</td><td></td></tr><tr><td>Avidin</td><td>Sigma</td><td>A9275</td><td></td></tr><tr><td>Streptavidin Beads</td><td>GE Healthcare&amp;nbsp;</td><td>17-5113-01</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Yeast tRNA</td><td>Sigma</td><td>R8508</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501</td><td></td></tr><tr><td>Yeast extract</td><td>Bacto</td><td>288620</td><td></td></tr><tr><td>peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>1x Phosphate-Buffered saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>0.2 M NaOH</td><td></td><td></td><td></td></tr><tr><td>4x Laemmli Sample Buffer (LSB)</td><td></td><td></td><td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td></tr><tr><td>Hot phenol lysis buffer</td><td></td><td></td><td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&amp;nbsp;</td></tr><tr><td>3 M Sodium Acetate pH 5.2</td><td></td><td></td><td></td></tr><tr><td>100% and 70% Ethanol (EtOH)</td><td></td><td></td><td></td></tr><tr><td>RNase-free water</td><td></td><td></td><td></td></tr><tr><td>RaPID lysis buffer</td><td></td><td></td><td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &amp;micro;g/ml Leupeptin, 10 &amp;micro;g/ml Aprotinin, 10 &amp;micro;g/ml Soybean Trypsin Inhibitor, 10 &amp;micro;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&amp;nbsp; Inhibitor.</td></tr><tr><td>2x Cross-linking reversal buffer</td><td></td><td></td><td>100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td></tr><tr><td>RaPID wash buffer</td><td></td><td></td><td>20 mM Tris-HCl pH 7.5,&amp;nbsp; 300 mM NaCl, 0.5% NP-40</td></tr><tr><td>0.5 M EDTA pH 8</td><td></td><td></td><td></td></tr><tr><td>Silver Stain Plus Kit</td><td>Bio-Rad&amp;nbsp;</td><td>161-0449</td><td>For detecting proteins in polyacrylamide gels</td></tr><tr><td>SD selective medium&amp;nbsp;</td><td></td><td></td><td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td></tr><tr><td>Anti-eEF3 (EF3A,YEF3)</td><td>Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td><td>1:5,000</td><td></td></tr><tr><td>Anti GFP antibody</td><td>Santa Cruz</td><td>sc-8334</td><td>1:3,000</td></tr><tr><td>Anti rabbit IgG-HRP conjugated</td><td>SIGMA</td><td>A9169</td><td>1:10,000</td></tr></tbody></table>

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.

RaPID enables the isolation of a specific target RNA with its associated proteins. Critical for its success is keeping the RNA intact as much as possible, thereby obtaining a sufficient amount of proteins. To determine the isolation efficiency and quality of RNA, northern analysis is performed (Figure 1A). Northern analysis has the advantage of directly reporting the efficiency and quality of RaPID. Thus, the relative amounts of full length and degradation products can be...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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 ...

novel-rna-binding-proteins-isolation-by-the-rapid-methodology

Research

JoVE Journal

Genetics

8.9K Views.  Technion - Israel Institute of Technology. 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. 

Video: Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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.

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. ...

Biochemistry

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 ...

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 ...

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 ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biology

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 ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

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

Source: Singh, R. <a href="https://app.jove.com/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.

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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