Name: Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions (Video) | JoVE Journal
Uploaded: 2017-09-28T14:00:00.000+00:00
Duration: 10 min 52 s
Description: Learn to identify RNA-binding proteins and map their binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry. This method requires minimal material and works without purification.

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5 minutes

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 replicates for the +4SU sample and the -4SU control. One 10-cm plate (i.e. 5 - 10 million cells) is more than sufficient for each biological replicate.</li>
	<li>The medium for mESCs is composed of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 0.5x penicillin/streptomycin, 1x non-essential amino acids, 100 &#181;M beta-mercaptoethanol, 1,000 U/mL leukemia inhibitory factor (LIF), 3 &#181;M CHIR99021, 1 &#181;M PD0325901.</li>
	<li>Before passaging mESCs, prepare the desired number of 10 cm plates.
	<ol>
		<li>Add 4 mL of 0.1% gelatin in water making sure that the entire surface of the plate is covered; incubate for 10 min at room temperature (RT); remove gelatin solution.</li>
	</ol>
	</li>
	<li>Passage cells when they reach ~10 million cells per plate, typically ~48 h following inoculation. 
	NOTE: As mESCs grow in dense colonies, plates should never be allowed to reach 100% confluency as observed for cell lines that grow in monolayers (e.g. HEK293 or 3T3 cells). An mESC plate should be passaged when ~50% confluent and definitely before the medium turns yellow.
	<ol>
		<li>To detach cells, wash plates with dense mESC cultures once in 1x phosphate buffered saline (PBS) supplemented with 2 mM EDTA, then add 0.05% trypsin dissolved in DMEM and return plate to incubator for 5 min (37 &#176;C, 5% CO2, and &#62;95% humidity).</li>
		<li>Remove plate from incubator and resuspend cells with equal volume of DMEM by pipetting up and down vigorously several times.</li>
		<li>Count cells using hemocytometer and ensure that cells form a single-cell suspension (i.e. no clusters), then centrifuge cell mixture 500 x g for 5 min at room temperature. Remove supernatant and resuspend cell pellet in 1 - 2 mL DMEM.</li>
		<li>Inoculate cells at a density of 10,000 cells/cm2 in mESC medium (i.e. ~800,000 cells per 10-cm plate) using the gelatin-coated plates prepared in step 1.3.1.</li>
	</ol>
	</li>
</ol>
2. Crosslink of Protein&#8211;RNA Interactions in Live Cells
NOTE: RNA-protein crosslinking is mediated by the photo-activatable ribonucleoside analog 4-thiouridine (4SU). 4SU has a longer absorbance maximum than endogenous nucleotides and can only be incorporated into RNA; therefore intermediate-wavelength UVB can be used to selectively crosslink RNA to proteins25,26. UVB treatment of 4SU-treated cells leads to covalent crosslinks between 4SU-containing RNA and amino acids, with a reported preference for Tyr, Trp, Met, Lys, and Cys27.
<ol>
	<li>Expand mESCs to the required number of 10-cm plates as described in step 1. 
	NOTE: Enough plates should be prepared for 3 - 5 biological replicates for the +4SU sample and the -4SU control. One 10-cm plate (i.e. 5 - 10 million cells) is sufficient for each biological replicate.</li>
	<li>Before the plates reach confluency, add 4SU directly into medium to a final concentration of 500 &#181;M. Leave -4SU control dishes untreated.</li>
	<li>Shake plates gently to distribute the 4SU homogeneously throughout the medium. Return them to the same tissue culture incubator for 2 h (37 &#176;C, 5% CO2, and &#62;95% humidity).</li>
	<li>Transfer plates to an ice bucket and discard medium.</li>
	<li>Rinse the plate gently with 5 mL of ice-cold PBS. 
	NOTE: Rinse gently or cells might detach.</li>
	<li>Add 2 mL of ice-cold PBS and place plates without lids in a crosslinker equipped with UVB-emitting bulbs (312 nm). Irradiate plates at an energy setting of 1 J/cm2. 
	NOTE: It is critical that plastic lids are removed or they might shield the cells from UV light and prevent crosslinking.</li>
	<li>Collect cells using cell lifters and transfer to ice-cold PBS in conical tubes. Centrifuge at 2,000 x g for 5 min, 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend cell pellet in 5 mL ice-cold PBS with 2 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride (PMSF).</li>
	<li>Count cells using a hemocytometer. Expect 5 - 10 and 10 - 20 million cells from 10-cm and 15-cm plates respectively.</li>
	<li>Centrifuge at 2,000 x g for 5 min, 4 &#176;C. 
	NOTE: Pelleted cells can be immediately used for step 3 or they can be flash-frozen in liquid nitrogen and stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
3. Isolation of Nuclei
NOTE: Nuclei are isolated to remove cytoplasmic proteins and increase coverage of nuclear proteins. This step can be replaced with other forms of cellular fractionation to study RBPs in different cellular compartments.
<ol>
	<li>Prepare ice cold Buffer A (10 mM Tris-HCl pH 7.9, 4 &#176;C, 1.5 mM MgCl2, 10 mM KCl). Use at least 2.5 mL per 10 million cells (as counted in step 2.9) for extraction. 
	NOTE: Stocks of Buffer A (e.g. a 500 mL bottle) can be prepared in advance and stored at 4 &#176;C for 6 months.
	<ol>
		<li>Prior to use, add 0.5 mM dithiothreitol (DTT) and 0.2 mM PMSF to the desired amount (2.5 mL per 10 million cells) of ice-cold buffer A.</li>
	</ol>
	</li>
	<li>Wash cells using 2 mL Buffer A per 10 million cells. Spin at 2,500 x g for 5 min at 4 &#176;C.</li>
	<li>Discard supernatant, add Buffer A supplemented with 0.2% of a nonionic, non-denaturing detergent, such as octyl-phenoxy-polyethoxy-ethanol (see the Table of Materials), 500 &#181;L per 10 million cells. Rotate for 5 min at 4 &#176;C.</li>
	<li>Centrifuge at 2,500 x g for 5 min.</li>
	<li>Remove the supernatant; the pellet comprises intact nuclei devoid of cytoplasm. 
	NOTE: Nuclei can be used immediately for step 4 or flash-frozen in liquid nitrogen and stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
4. Lysis of Nuclei
NOTE: Crosslinked nuclei are lysed in a mass spectrometry-compatible buffer to release proteins and protein-RNA complexes.
<ol>
	<li>Add lysis buffer (9 M urea, 100 mM Tris-HCl pH 8.0, 24 &#176;C), 200 &#181;L per 10 million cells. Sonicate to shear chromatin using a 1/8&#34; probe at 50% amplitude setting for 5 - 10 s.</li>
	<li>Spin at 12,000 x g for 5 min and collect 175 &#181;L of supernatant to avoid the pellet. 
	NOTE: 100 &#181;L will be used for the next RBR-ID steps and the remaining 75 &#181;L of lysate can be stored at -80 &#176;C for later use and/or western blot analysis.</li>
	<li>Measure protein concentration via Bradford assay or similar technique28. Dilute protein concentration in the different samples to 1 mg/mL or the lowest sample concentration using appropriate amounts of lysis buffer. 
	NOTE: From the nuclei of 10 million mESCs expect 100 -&#160;200 &#181;g of protein.</li>
</ol>
5. Trypsin Digestion
NOTE: Proteins are digested to generate peptides suitable for bottom-up mass spectrometry (MS) analysis.
<ol>
	<li>Add DTT solution to nuclear lysate (5 mM final); incubate for 1 h at RT.</li>
	<li>Add iodoacetamide from fresh stock (14 mM final); incubate for 30 min at RT in the dark.</li>
	<li>Dilute lysate with 5 times volume of 50 mM NH4HCO3</li>
	<li>Add trypsin (&#181;g protein : &#181;g trypsin = 200 : 1); incubate at 37 &#176;C overnight.</li>
</ol>
6. Desalting of peptides
<ol>
	<li>Prepare one custom-made stage tip per sample.
	<ol>
		<li>6.1.1. Place a disk of solid phase extraction C18 material into the bottom of a 200 &#181;L pipette tip.</li>
		<li>Add 50 &#181;L of oligo R3 reversed phase resin on top of the C18 disk. Place stage tip inside centrifugation adaptor and the place tip with adaptor inside a 1.5 mL microfuge tube.</li>
		<li>Add 100 &#181;L 100% acetonitrile to tips to wash. Centrifuge at 1000 x g for 1 min to remove the solvent.</li>
		<li>Equilibrate with 50 &#181;L 0.1% trifluoroacetic acid (TFA). Centrifuge at 1000 x g for 1 min.</li>
	</ol>
	</li>
	<li>Add 100% formic acid to digested protein sample to decrease the pH to 2 - 3. 
	NOTE: This should require ~5 &#181;L per 1 mL of diluted peptide sample, but pH should be measured and more formic acid added if necessary.</li>
	<li>Load sample onto custom-made stage tip, centrifuge at 1000 x g for 1 min until all of sample goes through the tip.</li>
	<li>Wash bound peptides with 50 &#181;L 0.1% TFA. Centrifuge at 1000 x g for 1 min.</li>
	<li>Elute peptides by adding 50 &#181;L elution buffer (75% acetonitrile, 0.025% TFA) to stage tip and centrifuge at 1000 x g for 1 min to force the elution buffer out of the tip.</li>
	<li>Dry the sample using a vacuum centrifuge set at 150 x g and 1 - 3 kPa for 1 h. 
	NOTE: Dried peptides can be stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
7. Removal of Crosslinked RNA
NOTE: Treat peptides with nuclease to remove crosslinked RNA.
<ol>
	<li>Resuspend the pellet in 50 &#181;L 2x nuclease buffer (100 mM NH4HCO3, 4 mM MgCl2).</li>
	<li>Measure peptide concentration using absorbance at 280 nm assuming 1 mg/mL of peptides for an A280 of 1. 
	NOTE: The expected concentration is 1 mg/mL.</li>
	<li>Adjust all samples to 1 mg/mL or to the lowest concentration using 2x nuclease buffer.</li>
	<li>Prepare a master mix of 50 &#181;L H2O + 1 &#181;L high purity nuclease (&#8805;250 U) (refer to the Table of Materials) per sample.</li>
	<li>Take 50 &#181;L of peptide sample and add 50 &#181;L of H2O + nuclease master mix.</li>
	<li>Incubate at 37 &#176;C for 1 h. Proceed to perform mass spectrometry. 
	NOTE: The peptides are now ready for mass spectrometry. They can be analyzed immediately or stored at -80 &#176;C indefinitely. This is a potential stopping point.</li>
</ol>
8. Nano Liquid Chromatography, Mass Spectrometry, and Raw Data Processing
NOTE: Because 4SU-crosslinking changes the mass of the peptide, their ions do not count toward the intensity of the non-crosslinked peptide during LC-MS/MS, which therefore appears to be decreased by the crosslinking. The degree and consistency of this decrease reflects the degree of protein-RNA crosslinking for each peptide24.
<ol>
	<li>Prepare HPLC buffers as follows: 0.1% formic acid in HPLC-grade water (buffer A); 0.1% formic acid in HPLC-grade acetonitrile (Buffer B).</li>
	<li>If a nano-HPLC is available, connect a nano-column with the following specifications: internal diameter 75 &#181;m; packed with C18-AQ particles; length 20 - 25 cm. 
	NOTE: If the HPLC available runs at higher flow rates use a proper column size for the indicated flow rate. Higher flow chromatography decreases sensitivity.</li>
	<li>Program the HPLC method as follows: flow rate 250 - 300 nL/min; gradient from 2 to 28% buffer B in 120 min, from 28 to 80% B for the next 5 min and isocratic 80% B for 10 min. 
	NOTE: If the HPLC does not have an automated column equilibration prior sample loading, start the program with 10 min at 0% B before loading the sample.</li>
	<li>Program the MS acquisition method to perform data-dependent acquisition (DDA) as standard shotgun proteomics experiments. The instrument duty cycle should alternate full MS scans with tandem MS scans (or MS/MS) of the most intense signals. Use the optimal settings for proteomics MS runs. 
	NOTE: For confident results, use instruments that can perform at least the full MS scan in high resolution mode (e.g. orbitrap, time-of-flight). For accurate quantification, make sure that intervals between full MS scans are no longer than 3 s. Longer duty cycles might prevent accurate definition of extracted ion chromatograms.</li>
	<li>Load 1 - 2 &#181;g of sample onto the HPLC column and run the HPLC-MS/MS method as programmed. For each biological replicate perform at least two independent runs and treat them as technical replicates.</li>
	<li>After MS, import the raw files into a proteomics software package for MS/MS spectra identification and peptide quantification via extracted ion chromatography. We recommend software packages that can perform chromatographic alignment of multiple MS runs (see the Table of Materials)29,30.</li>
	<li>If the option is available in the software suite, enable &#34;Match Between Runs&#34; prior to starting data processing.</li>
	<li>Once the analysis is finished, export the peptide list together with the quantification values.</li>
</ol>

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<li>MacLean, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skyline%3A+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li>
<li>Kondo, Y., Oubridge, C., van Roon, A. M., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+human+U1+snRNP%2C+a+small+nuclear+ribonucleoprotein+particle%2C+reveals+the+mechanism+of+5%27+splice+site+recognition%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.</a> Elife. 4, (2015).</li>
<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> J Vis Exp. (41), (2010).</li>
</ol>

The authors have nothing to disclose.

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&amp;times;)</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 &amp;micro;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

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...

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.

sample-preparation-for-mass-spectrometry-based-identification-rna

Research

JoVE Journal

Biochemistry

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.

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

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

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.

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

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

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.

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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.

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

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2'-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&lt;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Learn to identify RNA-binding proteins and map their binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry. This method requires minimal material and works without purification.

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

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