TITLE: Cell Lysis, Two-Step Purification, and RNase Digestion
AUTHORS: Clémentine Delan-Forino, David Tollervey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA in eukaryotes and Archaea. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but in most cases, it remains unclear how many substrates follow each pathway in vivo. Here we describe the method for using an UV cross-linking technique termed CRAC to generate stringent, transcriptome-wide mapping of exosome–substrate interaction sites in vivo and at base-pair resolution.</div><br/><div class = "text-block"><span>We present a protocol for the identification of RNA interaction sites for the exosome, using UV cross-linking and analysis of </span><span style = "font-style:italic;">c</span><span>DNA (CRAC) [</span><span style = ":UNDERLINE;">1</span><span>, </span><span style = ":UNDERLINE;">2</span><span>]. A number of related protocols for the identification of sites of RNA–protein interaction have been reported, including HITS-CLIP, CLIP-Seq, iCLIP, eCLIP, and others [</span><span style = ":UNDERLINE;">3</span><span>, </span><span style = ":UNDERLINE;">4</span><span>, </span><span style = ":UNDERLINE;">5</span><span>, </span><span style = ":UNDERLINE;">6</span><span>]. These all exploit protein immunoprecipitation to isolate protein–RNA complexes. CRAC is distinguished by the inclusion of tandem affinity purification and denaturing purification, allowing greater stringency in the recovery of authentic RNA–protein interaction sites.</span></div><br/><div class = "text-block"><span>To allow CRAC analyses, strains are created that express a “bait” protein with a tripartite tag. This generally consists of His6, followed by a TEV-protease cleavage site, then two copies of the z-domain from Protein A (HTP). The tag is inserted at the C terminus of the endogenous gene within the chromosome. The fusion construct is the only version of the protein expressed and this is under the control of the endogenous promoter. Several alternative tags have been successfully used, including a version with N-terminal fusion to a tag consisting of 3× FLAG-PreSission protease (PP) cleavage site-His6 (FPH) [</span><span style = ":UNDERLINE;">7</span><span>]. This is a smaller construct and is suitable for use on proteins with structures that are incompatible with C-terminal tagging. An additional variant is the insertion of a PP site into a protein that is also HTP tagged. This allows the separation of different domains of multidomain proteins. Importantly, the intact protein is cross-linked in the living cell, with domain separation in vitro. This has been successfully applied to the exosome subunit Rrp44/Dis3 to specifically identify binding sites for the PIN endonuclease domain [</span><span style = ":UNDERLINE;">8</span><span>].</span></div><br/><div class = "text-block"><span>Briefly, during standard CRAC analyses, covalently linked protein–exosome complexes are generated in vivo by irradiation with UV-C (254 nm). This generates RNA radicals that rapidly react with proteins in direct contact with the affected nucleotide (zero length cross-linking). The cells are then lysed and complexes with the bait protein are purified using an IgG column. Protein–RNA complexes are specifically eluted by TEV cleavage of the fusion protein and cross-linked RNAs trimmed using RNase A/T1, leaving a protected “footprint” of the protein binding site on the RNA. Trimmed complexes are denatured using 6 M Guanidinium, immobilized on Ni-NTA affinity resin and washed under denaturing conditions to dissociate copurifying proteins and complexes. The subsequent enzymatic steps are all performed on-column, during which RNA 3′ and 5′ ends are prepared, labeled with </span><span style = "vertical-align:super;">32</span><span>P (to allow RNA–protein complexes to be followed during gel separation) and linkers ligated. Note, however, that alternatives to using </span><span style = "vertical-align:super;">32</span><span>P labeling have been reported (e.g., [</span><span style = ":UNDERLINE;">6</span><span>]). The linker-ligated, RNA–protein complexes are eluted from the Ni-NTA resin and size selected on a denaturing SDS-PAGE gel. Following elution, the bound RNA is released by degradation of the bait protein using treatment with Proteinase K. The recovered RNA fragments are identified by reverse transcription, PCR amplification and sequencing using an Illumina platform.</span></div><br/><div class = "text-block"><span>Relative to CLIP-related protocols, CRAC offers the advantages of stringent purification, that substantially reduces background, and on-bead linker ligation that simplifies separation of reaction constituents during successive enzymatic steps. It also avoids the necessity to generate high-affinity antibodies needed for immunoprecipitation. Potential disadvantages are that, despite their ubiquitous use in yeast studies, tagged constructs may not be fully functional. This can be partially mitigated by confirming the ability of the tagged protein to support normal cell growth and/or RNA processing, or by comparing the behavior of N- and C-terminal tagged constructs. Additionally, because linkers are ligated to the protein–RNA complex, a possible disadvantage is that UV-cross-linking of the RNA at, or near, the 5′ or 3′ end it may sterically hinder on-column (de)phosphorylation and/or linker ligation. With these caveats, CRAC has been successfully applied to >50 proteins in budding yeast, and in other systems ranging from pathogenic bacteria to viral infected mouse cells [</span><span style = ":UNDERLINE;">7</span><span>, </span><span style = ":UNDERLINE;">9</span><span>].</span></div></div>

[STEPS]
?. [RNase Digestion and Binding to Ni-NTA Resin]
The concentration of RNaceIT used to footprint (trim) RNAs on protein of interest is determined empirically. Ideally, the reads will be long enough to map uniquely (~17 nt) but short enough to give good resolution of the protein-binding site. We aim to generate an average RNA length of ~30 nt. Commercially available RNase stock is highly concentrated, so to minimize discrepancies between experiments it is practical to prepare a working stock of RNases (use 1:100 dilution in water), store it at 4 °C and use for all subsequent experiments.
?. All steps must be carried out on ice, unless stated otherwise. For troubleshooting, it is a good idea to monitor the course of the experiment by retaining samples at points during the CRAC protocol. This allows potential problems with Protein–RNA purification steps to be identified. Three aliquots per sample are taken during the purification (Subheading 3.2.2 “Crude Lysate” and “IgG supernatant,” Subheading 3.2.3 “TEV Eluate”). These can be analyzed by Western blot.
?. [Cell Lysis]
Add one volume () of TN150 lysis buffer (+ protease inhibitors) to cell pellets and thaw rapidly.
1 mL
1 L of cell culture yields pellets of approximately 1 ml, but lysis volumes should be increased if larger cultures were used.
?. [Cell Lysis]
Add 2.5 volumes of Zirconia beads to cell pellet (~ , measure in 15 ml Falcon tubes). Vortex the cells , then put them  for   between each vortexing step. Repeat five more times.
2.5 mL
on ice
Using a powerful vortex increases the efficiency of the lysis.
?. [Cell Lysis]
Add three volumes of TN150 (+ protease inhibitors) (~), shake vigorously, and centrifuge the suspension in the falcon tube ().
3 mL
Centrifuge: 4000 34, 20 min, 4 10
?. [Cell Lysis]
Transfer the supernatant (~ ) to three 1.5 ml tubes and spin lysate at  in a microcentrifuge. Keep tubes  throughout the process.
4.5 mL
Centrifuge: 16000 34, 20 min, 4 10
on ice
?. [Cell Lysis]
Keep supernatants , ready to be mixed with IgG sepharose beads in Step 10 below.
on ice
?. [Protein A:IgG Purification]
Resuspend IgG Sepharose beads by gently inverting the bottle. Pipette out  into a 15 ml tube. The bead:buffer ratio is approximately 75:25 so this equates to 125 μl of beads. Wash the beads twice with . Collect beads between washes by pulsing in the centrifuge to  ().
[stock per sample]
[TN150 lysis buffer + protease inhibitors]
Centrifuge: 1000 33
Centrifuge: 210 34
Use wide orifice pipette tips or use a sterile scalpel to cut the end off of a regular pipette tip for more efficient pipetting of the beads.
?. [Protein A:IgG Purification]
Remove the supernatant after the second wash and add  per sample. Gently resuspend the beads, and aliquot  per sample into 15 ml tubes.
[TN150 + protease inhibitors]
[mix]
?. [Protein A:IgG Purification]
Take  aliquots for troubleshooting the purification, and store at .
[cleared lysate ("crude lysate")]
-20 °C
?. [Protein A:IgG Purification]
Mix remaining lysates with IgG Sepharose beads and rotate for a minimum of  at . This step can be extended to .
4 °C
?. [Protein A:IgG Purification]
Collect the beads by pulsing to   and remove most of the supernatant.
Centrifuge: 1000 33, 4 10
?. [Protein A:IgG Purification]
Take a  aliquot for troubleshooting (“IgG supernatant”) and store at .
[supernatant]
-20 °C
?. [Protein A:IgG Purification]
Wash beads twice with  and twice with  For each wash, gently agitate at  for .
[TN1000]
[TN150]
4 °C
Buffers used in this and all subsequent steps should not contain protease inhibitors.
?. [Protein A:IgG Purification]
Gently resuspend gently the beads in  and transfer to a 1.5 ml tube.
[TN150]
?. [TEV Cleavage]
Add – of TEV protease to the beads and mix by inverting tube.
20
30
?. [TEV Cleavage]
Incubate at  for  with shaking (make sure beads remain in suspension).
18 °C
?. [TEV Cleavage]
Pass the mixture through a microcentrifuge column (SnapCap) to remove the beads. Spin column () to collect all the eluate in a 1.5 ml tube. Do not put eluate on ice.
Centrifuge: 1000 33
?. [TEV Cleavage]
Take a  (“TEV eluate”) and store at .
[eluate sample]
-20 °C
?. [RNase Digestion and Binding to Ni-NTA Resin]
Prepare 1.5 ml microcentrifuge tubes containing .
[ guanidine HCl]
?. [RNase Digestion and Binding to Ni-NTA Resin]
Prepare  by pipetting  into tubes and wash them twice with . Leave the resin in about  until use.
[Ni-NTA resin]
[50% slurry]
[Wash Buffer I]
[Wash Buffer I]
?. [RNase Digestion and Binding to Ni-NTA Resin]
Add  (0.15 units, Agilent) to . Incubate for  at .
[1:100 dilution of RNace IT]
[TEV eluate]
37 °C
?. [RNase Digestion and Binding to Ni-NTA Resin]
Immediately transfer the RNase treated eluate into the prepared microcentrifuge tubes containing guanidine hydrochloride. The final volume will be around .
700 µl
?. [RNase Digestion and Binding to Ni-NTA Resin]
Add  ( final concentration) and  (final concentration) to the eluate.
[5 M NaCl]
[2.5 M pH 8.0 imidazole ]
?. [RNase Digestion and Binding to Ni-NTA Resin]
Remove the remaining supernatant from the washed Ni-NTA resin. Add the eluate to the resin.
?. [RNase Digestion and Binding to Ni-NTA Resin]
Incubate with gentle agitation at .
4 °C
?. [RNase Digestion and Binding to Ni-NTA Resin]
Pulse the microcentrifuge tubes to collect the resin. Remove the supernatant and wash three times with  and three times with .
[Wash Buffer I]
[1× PNK buffer]
?. [RNase Digestion and Binding to Ni-NTA Resin]
Transfer the resin to a microcentrifuge column (SnapCap).
?. [RNase Digestion and Binding to Ni-NTA Resin]
Western blot with “Crude Lysate,” “IgG supernatant,” and “Tev Eluate” controls can be carried out in parallel of the steps below (Fig. 1a). If needed, additional controls can be prepared by taking aliquots of the experiments in other steps, such as pipetting out 30 μl of IgG beads before addition of TEV protease and 30 μl after TEV treatment to control binding and cleavage efficiency.