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 border="0" cellpadding="0" cellspacing="0" width="632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tris</td>
			<td style="width:107px;">sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>bio-lab</td>
			<td align="right">1981232300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol (pH 4.3)</td>
			<td>sigma</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol: Chloroform (5:1, pH 4.3)</td>
			<td>sigma</td>
			<td>P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>bio-lab</td>
			<td align="right">3080521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Frutarom</td>
			<td align="right">5551820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>sigma</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Calbiochem</td>
			<td align="right">492016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl Flouride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonuclease&#160; Inhibitor</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Beads</td>
			<td>Sartorius</td>
			<td>BBI-8541701</td>
			<td>0.4-0.6mm diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini BeadBeater</td>
			<td>BioSpec</td>
			<td colspan="2">Mini BeadBeater 16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium</td>
			<td>Sigma</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin</td>
			<td>Sigma</td>
			<td>A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads</td>
			<td>GE Healthcare&#160;</td>
			<td>17-5113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA</td>
			<td>Sigma</td>
			<td>R8508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Bacto</td>
			<td dir="LTR">288620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Bacto</td>
			<td dir="LTR">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 x Phosphate-Buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 x 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 height="21">
			<td height="21" style="height:21px;">Hot phenol lysis buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 M Sodium Acetate pH 5.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% and 70% Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RaPID lysis buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &#181;g/ml Leupeptin, 10 &#181;g/ml Aprotinin, 10 &#181;g/ml Soybean Trypsin Inhibitor, 10 &#181;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&#160; Inhibitor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Cross-linking reversal buffer</td>
			<td></td>
			<td></td>
			<td dir="LTR">100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RaPID wash buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris-HCl pH 7.5,&#160; 300 mM NaCl, 0.5% NP-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EDTA pH 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Stain Plus Kit</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0449</td>
			<td>For detecting proteins in polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SD selective medium&#160;</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 height="21">
			<td height="21" style="height:21px;">Anti-eEF3 (EF3A,YEF3)</td>
			<td colspan="2">Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td>
			<td>1:5,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti GFP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8334</td>
			<td>1:3,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

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 ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...