TITLE: Linker Ligation at both Ends of RNAs on Beads
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]
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
TSAP catalyzes removal of 5′ and 3′ phosphate groups from DNA and RNA; it is effective on 3′ overhangs, 5′ overhangs and blunt ends and leaves 5′ OH and 3′ OH ends. Treating the RNAs with alkaline phosphatase will remove the 3′ phosphates left behind by the RNase cleavage of the RNA.
?. [On Bead Ligation of 3′ miRCat-33 Linker]
The 3′-linker is a DNA oligonucleotide that has a blocked 3′ end to prevent self-ligation and a 5′-end that is preactivated by adenylation (AppN…). T4 RNA ligase usually activates its substrate by preadenylation using ATP. Employing a preadenylated linker allows the reactions to be performed in the absence of ATP. This decreases the risk of circularizing any remaining 5′-phosphorylated RNA; a side reaction that would otherwise be expected. Moreover, addition of ATP in the mix could inhibit the reaction, as the active site of T4 RNA ligase would get adenylated and could not transfer the adenosine to any substrate as the linker is already adenylated.
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
The labeling reaction is performed first with radioactive ATP only to obtain a reasonable radioactive signal from the RNA in the sample. Subsequently, nonlabeled ATP is added to the reaction to allow efficient phosphorylation of all RNA 5′ ends in the sample, required for 5′ end adapter ligation.
?. [On-Column Ligation of the 5′ Adapters]
These linkers have blocked 5′ end to prevent self-concatenation. Moreover, they contain barcodes allowing distinction of samples in case of multiplexing and random nucleotides to distinguish molecules with same 5′- and 3′-end (allowing removal of PCR duplicates). It is crucial to use different barcodes for each sample.
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
Spin out the residual 1× PNK buffer and close the column with the supplied press-on bottom stopper.
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
Incubate at  for .
37 °C
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
Wash the resin once with  and three times with .
[Wash Buffer I]
[1× PNK buffer]
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
To each sample, add  that contains:
[TSAP master mix]
[5× PNK buffer]
[TSAP]
[RNasin]
[Milli-Q water]
?. [Dephosphorylation of RNA 3′ P Ends Using Alkaline Phosphatase (TSAP)]
Mix by stirring with a pipette tip then flicking column gently.
?. [On Bead Ligation of 3′ miRCat-33 Linker]
Spin out the residual volume of 1× PNK buffer. Close the bottom of the column with the press-on stopper.
?. [On Bead Ligation of 3′ miRCat-33 Linker]
To each sample add .
[T4 RNA ligase I]
?. [On Bead Ligation of 3′ miRCat-33 Linker]
Incubate at  for .
25 °C
?. [On Bead Ligation of 3′ miRCat-33 Linker]
Wash the resin once with  and three times with .
[Wash Buffer I]
[1× PNK buffer]
?. [On Bead Ligation of 3′ miRCat-33 Linker]
To each sample, add   containing:
[miRCat master mix]
[5× PNK buffer]
[10 μM miRCat-33]
[RNasin]
[Milli-Q water]
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
Spin out the residual volume of 1× PNK buffer. Cap the bottom of the column.
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
Add  and continue the incubation for .
[100 mM ATP]
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
Wash the resin four times with  and three times with .
[Wash Buffer I]
[1× PNK buffer]
Additional washes can be done to remove most free radioactive ATP and decrease the chance of radioactive contamination at later stages. Perform the washes until the radioactivity of the flow through measured with a manual Geiger counter falls to approximately 10–15 cps.
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
To the resin, add  containing:
[PNK master mix]
[5× PNK buffer]
[T4 polynucleotide kinase]
[Milli-Q water]
[ 32P-ATP (10 μCi/μl)]
?. [Phosphorylating the 5′ Ends of Cross-Linked RNA]
Incubate at  for .
37 °C
?. [On-Column Ligation of the 5′ Adapters]
Spin out the residual volume of 1× PNK buffer.
?. [On-Column Ligation of the 5′ Adapters]
To each sample add  and .
[barcoded 5′ linker ]
[T4 RNA ligase I]
?. [On-Column Ligation of the 5′ Adapters]
Incubate  at .
16 °C
?. [On-Column Ligation of the 5′ Adapters]
Wash the resin three times with Wash Buffer II.
?. [On-Column Ligation of the 5′ Adapters]
Add  containing:
[5′ linker master mix ]
[5× PNK]
[10 mM ATP]
[RNasin]
[Milli-Q water]