TITLE: Cell Culture and UV Cross-Linking
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 cDNA (CRAC) [1, </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>, 4, </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]
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Streak out HTP-tagged and negative control strains from glycerol stocks onto YPD plates (or SD medium—leucine when required for plasmid maintenance) and incubate at  for –.
30 °C
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Inoculate single colonies into  (YNB−amino acids, CSM−TRP, and 2% glucose) and incubate at  with shaking at  for , then use to inoculate .
[SD medium]
30 °C
33
[overnight cultures]
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Inoculate  with the quantity of overnight culture required to obtain an OD600 around 0.05, and grow until the OD600 measurement reaches 0.5.
[prewarmed minimal media]
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Clean the UV cross-linker with deionized water before use, and prewarm the lamp.
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Irradiate cultures in the UV cross-linker at  for  (254 nm, equivalent to an average dose of ∼1.6 mJ cm−2).
30 Room temperature
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Pour the cultures into prechilled 1L centrifuge bottles . Harvest by centrifugation; .
on ice
Centrifuge: 2700 34, 15 min, 4 10
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Resuspend pellet in  and transfer to a 50 ml Falcon tube. Centrifuge for .
[ice-cold phosphate-buffered saline (PBS) ]
Centrifuge: 3000 34, 5 min, 4 10
We usually keep the three pellets in individual tubes and use one per CRAC experiment.
?. [Growth and Cross-Linking of RNA–Exosome Subunit-His-TEV-ProteinA (HTP) Complexes]
Discard supernatant and store pellets at  until required.
-80 °C