R.B. was supported by the Searle Scholars Program, the W.W. Smith Foundation (C1404), and the March of Dimes Foundation (1-FY-15- 344). B.A.G acknowledges support from NIH grants R01GM110174 and NIH R01AI118891, as well as DOD grant BC123187P1. R.W.-T. was supported by NIH training grant T32GM008216.

1. Culture and Expansion of mESCs
NOTE: Mouse embryonic stem cells are easy to culture and can be quickly expanded to the large numbers required by biochemical experiments thanks to their fast cycling time. Healthy mESCs double every 12 h.
<ol>
	<li>Expand mESCs to the desired number on gelatinized plates in mESC medium (see below) in a tissue culture incubator kept at 37 &#176;C, 5% CO2, and &#62;95% humidity. 
	NOTE: Enough plates should be prepared for 3 - 5 biological rep...

<ol>
	<li>Bonasio, R., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+transcription+by+long+noncoding+RNAs.">Regulation of transcription by long noncoding RNAs.</a> Annu Rev Genet. 48, 433-455 (2014).</li><li>Bonasio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+topics+in+epigenetics:+ants,+brains,+and+noncoding+RNAs.">Emerging topics in epigenetics: ants, brains, and noncoding RNAs.</a> Ann N Y Acad Sci. 1260, 14-23 (2012).</li><li>Rinn, J. L., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+regulation+by+long+noncoding+RNAs.">Genome regulation by long noncoding RNAs.</a> Annu Rev Biochem. 81, 145-166 (2012).</li><li>Goff, L. A., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+RNA+biology+to+lncRNAs.">Linking RNA biology to lncRNAs.</a> Genome Res. 25 (10), 1456-1465 (2015).</li><li>Holoch, D., Moazed, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+epigenetic+regulation+of+gene+expression.">RNA-mediated epigenetic regulation of gene expression.</a> Nat Rev Genet. 16 (2), 71-84 (2015).</li><li>Chu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+discovery+of+Xist+RNA+binding+proteins.">Systematic discovery of Xist RNA binding proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>McHugh, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li><li>Minajigi, 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=Chromosomes.+A+comprehensive+Xist+interactome+reveals+cohesin+repulsion+and+an+RNA-directed+chromosome+conformation.">Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.</a> Science. 349 (6245), (2015).</li><li>Lunde, B. M., Moore, C., Varani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins:+modular+design+for+efficient+function.">RNA-binding proteins: modular design for efficient function.</a> Nat Rev Mol Cell Biol. 8 (6), 479-490 (2007).</li><li>Cabili, M. N., 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=Integrative+annotation+of+human+large+intergenic+noncoding+RNAs+reveals+global+properties+and+specific+subclasses.">Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses.</a> Genes Dev. 25 (18), 1915-1927 (2011).</li><li>Castello, 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=Insights+into+RNA+biology+from+an+atlas+of+mammalian+mRNA-binding+proteins.">Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.</a> Cell. 149 (6), 1393-1406 (2012).</li><li>Baltz, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mRNA-bound+proteome+and+its+global+occupancy+profile+on+protein-coding+transcripts.">The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.</a> Mol Cell. 46 (5), 674-690 (2012).</li><li>Kwon, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+protein+repertoire+of+embryonic+stem+cells.">The RNA-binding protein repertoire of embryonic stem cells.</a> Nat Struct Mol Biol. 20 (9), 1122-1130 (2013).</li><li>Beckmann, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA-binding+proteomes+from+yeast+to+man+harbour+conserved+enigmRBPs.">The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.</a> Nat Commun. 6, 10127 (2015).</li><li>Conrad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+interactome+capture+of+the+human+cell+nucleus.">Serial interactome capture of the human cell nucleus.</a> Nat Commun. 7, 11212 (2016).</li><li>Wilusz, J. 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=A+triple+helix+stabilizes+the+3'+ends+of+long+noncoding+RNAs+that+lack+poly(A)+tails.">A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.</a> Genes Dev. 26 (21), 2392-2407 (2012).</li><li>Kim, T. K., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectural+and+Functional+Commonalities+between+Enhancers+and+Promoters.">Architectural and Functional Commonalities between Enhancers and Promoters.</a> Cell. 162 (5), 948-959 (2015).</li><li>Brannan, K. W., 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=SONAR+Discovers+RNA-Binding+Proteins+from+Analysis+of+Large-Scale+Protein-Protein+Interactomes.">SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.</a> Mol Cell. 64 (2), 282-293 (2016).</li><li>Bonasio, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+with+RNA+direct+the+Polycomb+group+protein+SCML2+to+chromatin+where+it+represses+target+genes.">Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.</a> Elife. 3, e02637 (2014).</li><li>Kaneko, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+JARID2+and+noncoding+RNAs+regulate+PRC2+recruitment+to+chromatin.">Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.</a> Mol Cell. 53 (2), 290-300 (2014).</li><li>Kaneko, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+the+PRC2+component+Ezh2+is+cell+cycle-regulated+and+up-regulates+its+binding+to+ncRNA.">Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.</a> Genes Dev. 24 (23), 2615-2620 (2010).</li><li>Saldana-Meyer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTCF+regulates+the+human+p53+gene+through+direct+interaction+with+its+natural+antisense+transcript,+Wrap53.">CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.</a> Genes Dev. 28 (7), 723-734 (2014).</li><li>Castello, 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=Comprehensive+Identification+of+RNA-Binding+Domains+in+Human+Cells.">Comprehensive Identification of RNA-Binding Domains in Human Cells.</a> Mol Cell. 63 (4), 696-710 (2016).</li><li>He, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Mapping+of+RNA-Binding+Regions+in+the+Nuclear+Proteome+of+Embryonic+Stem+Cells.">High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.</a> Mol Cell. 64 (2), 416-430 (2016).</li><li>Favre, 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=4-Thiouridine+photosensitized+RNA-protein+crosslinking+in+mammalian+cells.">4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.</a> Biochem Biophys Res Commun. 141 (2), 847-854 (1986).</li><li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP.">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li><li>Favre, A., Saintome, C., Fourrey, J. L., Clivio, P., Laugaa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thionucleobases+as+intrinsic+photoaffinity+probes+of+nucleic+acid+structure+and+nucleic+acid-protein+interactions.">Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.</a> J Photochem Photobiol B. 42 (2), 109-124 (1998).</li><li>Bradford, M. 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We describe a detailed experimental protocol to perform RBR-ID in mESCs and, with appropriate modifications, in any cell that can incorporate 4SU into RNA. Other cell types may require optimization of the approach to ensure a sufficient signal to noise ratio. Additionally, while the protocol described herein focuses on the examination of nuclear RBPs, the RBR-ID technology should be easily adapted to different cellular compartments, such as the cytosol or specific organelles, by use of different fractionation strategies....

The purpose of the RBR-ID method is to identify novel RNA-binding proteins (RBPs) and map their RNA-binding regions (RBRs) with peptide-level resolution to facilitate the design of RNA-binding mutants and the investigation of the biological and biochemical functions of protein-RNA interactions.
RNA is unique among biomolecules as it can both act as a messenger carrying genetic information and also fold into complex three-dimensional structures with biochemical functions more akin to those of proteins1,2. A growing body of evidence suggests that noncoding RNAs (ncRNAs) play important roles in various gene regulatory and epigenetic pathways3,4,5 and, typically, these regulatory functions are mediated in concert with proteins that interact specifically with a given RNA. Of particular relevance, a set of interacting proteins was recently identified for the intensely studied long ncRNA (lncRNA) Xist, providing valuable insight into how this lncRNA mediates X-chromosome inactivation in female cells6,7,8. Notably, several of these Xist-interacting proteins do not contain any canonical RNA-binding domains9, and therefore their RNA-binding activity could not be predicted in silico based on their primary sequence alone. Considering that thousands of lncRNAs are expressed in any given cell10, it is reasonable to assume that many of them might act via interactions with yet to be discovered RNA-binding proteins (RBPs). An experimental strategy to identify these novel RBPs would therefore greatly facilitate the task of dissecting the biological function of ncRNAs.
Previous attempts to identify RBPs empirically have relied on polyA+ RNA selection coupled to mass spectrometry (MS)11,12,13,14,15. Although these experiments added many proteins to the list of putative RBPs, by design they could only detect proteins bound to polyadenylated transcripts. However, most small RNAs and many lncRNAs are not polyadenylated.16,17 and their interacting proteins would likely have been missed in these experiments. A recent study applied machine learning to protein-protein interactome databases to identify proteins that co-purified with multiple known RBPs and showed that these recurrent RBP partners were more likely to possess RNA-binding activities18. However, this approach relies on mining existing large interaction databases and can only identify proteins that can be co-purified in non-denaturing conditions with known RBPs, thus excluding from the analysis insoluble, membrane-embedded, and scarce proteins.
The identification of a protein as a bona fide RBP often does not automatically yield information on the biological and/or biochemical function of the protein-RNA interaction. To address this point, it is typically desirable to identify the protein domain and amino acid residues involved in the interaction so that specific mutants can be designed to test the function of RNA binding in the context of each novel RBP19,20. Previous efforts by our group and others have used recombinant protein fragments and deletion mutants to identify RNA binding regions (RBRs)19,20,21,22; however, such approaches are labor-intensive and incompatible with high-throughput analyses. More recently, a study described an experimental strategy to map RNA-binding activities in a high-throughput fashion using mass spectrometry23; however, this approach relied on a double polyA+ RNA selection, and thus carried the same limitations as the RBP identification approaches described above.
We developed a technique, termed RNA binding region identification (RBR-ID), which exploits protein-RNA photocrosslinking and quantitative mass spectrometry to identify proteins and protein regions interacting with RNA in live cells without making assumption on the RNA polyadenylation status, thus including RBPs bound to polyA- RNAs24. Moreover, this method relies exclusively on crosslinking and has no requirements on protein solubility or accessibility and is thus suitable to map RNA-binding activities within membranes (e.g. the nuclear envelope) or poorly soluble compartments (e.g. the nuclear matrix). We describe the experimental steps to perform RBR-ID for the nuclei of mouse embryonic stem cells (mESCs) but with minor modifications this protocol should be suitable for a variety of cell types, provided that they can efficiently incorporate 4SU from the culture medium.

<table>
	<tbody>
		<tr>
			<td>KnockOut DMEM</td>
			<td>Fisher Scientific</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, qualified, US origin</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution 200 mM</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor (LIF)</td>
			<td>EMD Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Sigma</td>
			<td>PZ0162</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin solution,2% in water</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>4-thiouridine</td>
			<td>Sigma</td>
			<td>T4509</td>
			<td>50 mM stock in water</td>
		</tr>
		<tr>
			<td>Spectrolinker XL-1500</td>
			<td>Fisher Scientific</td>
			<td>11-992-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma</td>
			<td>78830</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CO-630</td>
			<td>Sigma</td>
			<td>542334</td>
			<td>Commercial form of octyl-phenoxy-polyethoxy-ethanol detergent</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
		<tr>
			<td>Empore solid phase extraction disk</td>
			<td>3M</td>
			<td>66883</td>
			<td></td>
		</tr>
		<tr>
			<td>OLIGO R3 Reversed - Phase Resin</td>
			<td>Fisher Scientific</td>
			<td>1133903</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma</td>
			<td>E8263</td>
			<td>High purity nuclease</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fisher Scientific</td>
			<td>discontinued</td>
			<td>updated with FB505110</td>
		</tr>
		<tr>
			<td>HPLC grade acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>W6 4</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Fisher Scientific</td>
			<td>A11650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma</td>
			<td>49199</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur 18-AQ</td>
			<td>Dr. Maisch GmbH HPLC</td>
			<td>r13.aq.0003</td>
			<td>Packing material for HPLC column</td>
		</tr>
		<tr>
			<td>Capillary for nano columns (75 &#181;m)</td>
			<td>Molex</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant software</td>
			<td>Max Planck Institute for Biochemistry</td>
			<td></td>
			<td>Can perform chromatographic alignment of multiple MS runs</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 depicts the RBR-ID workflow. Due to the relatively low crosslinking efficiency of this technique, it is very important to consider both the depletion level and consistency of the observed effect (P-value) across biological replicates. Figure 2 shows a volcano plot of RBR-ID result. Peptides that overlapped RNA recognition motif (RRM) domain show highly consistent depletion level. RRM domains can be used as a pos...

Watch this Scientific Journal Video about Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions at JoVE.com

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

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 ...

The overall goal of this procedure is to identify RNA binding proteins in live cells and map their RNA binding regions using UV mediated photo cross-linking followed by mass spectrometry. This method can help answer key questions in the field of epigenetics, such as which epigenetic regulators are bound to RNA and what protein regions are required for the interactions. The main advantage of this technique is that it does not involve purification of the protein RNA complexes, and therefore requires little material and identifies proteins bound to any type of RNA.As presented, this method focuses on nuclear proteins because that is our scientific interest, but with minor modifications in the fractionation steps, it could be used to study RNA and binding proteins in other stu-nu-lar compartments. After culturing and expanding mouse ESCs according to the text protocol, expand the cells to the required number of 10 centimeter plates. Before the plates reach confluency, add four SU directly into the medium to a final concentration of 500 micro molar.Leave 4sU control dishes untreated. Gently shake the plates to homogeneously distribute the 4sU throughout the medium, then return the plates to the same tissue culture incubator for two hours. The efficiency of 4sU incorporation varies across cell types, and therefore it might be necessary to optimize it's concentration and incubation time to ensure efficient cross-linking and minimize toxicity.Next, transfer the plates to an ice bucket and discard the medium. Then, use 5 milliliters of ice-cold PBS to gently rinse the plates. Add 2 milliliters of ice-cold PBS and place the plates without lids into a cross-linker equipped with UV-B emitting bulbs.Irradiate the plates at an energy setting of 1 Joule per square centimeter. Efficient UV cross-linking is critical. We recommend using UV-B light, but UV-A can also be used.In any case, the amount of UV energy used should be optimized and can be monitored by PAR-CLIP or ChIRP. Use cell lifters to collect the cells and transfer them to ice-cold PBS in conical tubes. Then, centrifuge the cells at 2000 times g and four degrees celsius for five minutes.Remove the supernatant and re-suspend the cell pellet in five milliliters of ice-cold PBS with 2 millimolar EDTA and 0.2 millimolar PMSF. With a hemocytometer, count the cells. Expect five to ten, and ten to twenty million cells from 10 centimeter and 15 centimeter plates, respectively.Then, centrifuge the cells at 2000 times g and 4 degrees celsius for five minutes. To isolate nuclei from the cells, prepare ice-cold buffer A as outlined in the text protocol. Prior to use, add 0.5 millimolar DTT and 0.2 millimolar PMSF to the desired amount of buffer A.Add 2 milliliters of the cold buffer to the cells to wash them, then spin the cells at 2500 times g and four degrees celsius for five minutes.Discard the supernatant, and add buffer A supplemented with 0.2 percent of a non-ionic, non-denaturing detergent, then rotate the sample at 4 degrees celsius for five minutes. After spinning the cells at 2500 times g for five minutes, remove the supernatant. The pellet comprises intact nuclei devoid of cytoplasm.To lyse the nuclei, add 200 microliters of lysis buffer to the tube. Use a 1/8th inch probe to sonicate the sample to shear the chromatin at a 50 percent amplitude setting for five to ten seconds. Spin the sample at 12000 times g for five minutes and collect only 175 microliters of supernatant to avoid the pellet.Next, measure the protein concentration via the Bradford assay or similar technique. Then, use lysis buffer to dilute the protein in the different samples to 1 milligram per milliliter or the lowest sample concentration. Add 5 millimolar DTT solution to the nuclear lysate and incubate the sample at room temperature for one hour.Then, add 14 millimolar iodoacetamide from fresh stock and incubate the lysate at room temperature in the dark for 30 minutes. Dilute the lysate with five times the volume of 50 millimolar ammonium bicarbonate, then add trypsin. Incubate the sample at 37 degrees celsius overnight.To prepare one custom-made stage tip per sample, place a disk of solid phase extraction C18 material into the bottom of a 200 microliter pipette tip. Add 50 microliters of Oligo R3 reverse-phased resin on top of the C18 disk, then place the stage tip inside a centrifugation adapter and place the adapter inside a 1.5 milliliter microfuge tube. Add 100 microliters of 100 percent acetonitrile to the tips to wash them.Then centrifuge the tips at 1000 times g for one minute to remove the solvent. Equilibrate the tips with 50 microliters of 0.1 percent trifluoroacetic acid or TFA. Then, spin them again.Add 100 percent formic acid to the digested protein sample to decrease the PH to 2 to 3. Then, load the sample onto a custom-made stage tip and centrifuge the sample at 1000 times g for one minute until all of the sample goes through the tip. Wash the bound peptides with 50 microliters of 0.1 percent TFA.Then, after spinning the sample at 1000 times g for one minute, elute the peptides by adding 50 microliters of elution buffer to the stage tip and centrifuge at 1000 times g for one minute to force the elution buffer out of the tip. Use a vacuum centrifuge set at 150 times g and 1 to 3 kilopascals for one hour to dry the sample. To remove cross-linked RNA from the sample, re-suspend the pellet in 50 microliters of 2x nuclease buffer.Measure the peptide concentration at 280 nanometers assuming 1 milligram per milliliter of peptides for and A280 of 1. Use 2x nuclease buffer to adjust all samples to one milligram per milliliter or to the lowest concentration, then prepare a master mix of 50 microliters of water plus 1 microliter of high purity nuclease per sample. Combine 50 microliters of peptide sample with 50 microliters of water plus nuclease master mix.Then, incubate the sample at 37 degrees celsius for one hour. Finally, carry out nanoliquid chromatography, mass spectrometry, and data processing according to the text protocol. This figure shows a volcano plot of an RBR id result from mouse ESCs.Peptides that overlapped with an RNA recognition motif domain are in blue and show highly consistent depletion levels. An RNA binding peptide from HNRNPC is highlighted in red. Shown here is a heat map of RBR id scores that are calculated as an estimate of a protein region's RNA binding likelihood.The score is projected on the surface of the spliceosomal subunit U1-70k. The bright red color in the vicinity of the RNA contact, as determined by the crystal structure, indicates correct identification of the protein RNA interaction. In this figure, TET2 is presented as a novel RBP and the RNA binding activity is mapped to a C terminal region by plotting the RBR id score along the primary sequence of the protein.Finally, the requirement for this novel RBR could be verified by performing PAR-CLIP using the wild type sequence and comparing the signal to that of a mutant lacking the predicted RBR. Once mastered, this technique can be done in seven to eight hours over two days if it is performed properly. Prior to cross-linking, cells can be stimulated with various treatments in order to answer additional questions like how does differentiation start or how does the cell cycle regulate RNA protein interactions.After watching this video, you should have a good understanding of how to generate extras containing cross-linked protein RNA complexes to identify sites of protein RNA interactions with mass spectrometry.

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JoVE Journal

Biochemistry

8.0K visualizações.  University of Pennsylvania Perelman School of Medicine. The overall goal of this procedure is to identify RNA binding proteins in live cells and map their RNA binding regions using UV mediated photo cross-linking followed by mass spectrometry. This method can help answer key questions in the field of epigenetics, such as which epigenetic regulators are bound to RNA and what protein regions are required for the interactions. The main advantage of this technique is that it does not involve purification of the protein RNA complexes, and therefore req...