Name: par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins
Uploaded: 2010-07-02T17:00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

We thank members of the Tuschl laboratory for helpful discussions. M.H. is supported by the Deutscher Akademischer Austauschdienst (DAAD). This work was supported by the Swiss National Fund Grant #3100A0-114001 to M.Z.; T.T. is an HHMI investigator, and work in his laboratory was supported by NIH grants GM073047 and MH08442 and the Starr Foundation.

The protocol below describes the PAR-CliP procedure for HEK293 cells expressing FLAG/HA-tagged IGF2BP1 upon induction with doxycycline. We will use an anti-FLAG antibody for immunoprecipitation.
PAR-CliP will work with any cell line expressing detectable levels of the endogenous, untagged RNA binding protein (RBP) of interest if an efficient antibody for immunoprecipitation is available.
Expanding Cells
<ol>
	<li>Expand FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 cells in growth medium. We recommend using between 100-400 x 106 cells (approx. 10-40 15 cm cell culture plates) as a starting point. Grow them to approximately 80% confluency.</li>
 <li>14 h before crosslinking add a) 4-thiouridine to a final concentration of 100 &mu;M (1:1000 v/v of a 1 M 4-thiouridine stock solution) directly to the cell culture medium and b) induce expression of the FLAG/HA tagged IGF2BP1 by addition of 1 &mu;g/ml of doxycycline (1:10,000 v/v of 10 mg/ml doxycycline stock solution). NOTE: instead of 4-thiouridine you can also use 100 &mu;M of 6-thioguanosine.</li>
</ol>
UV-Crosslinking
<ol>
	<li>Wash cells once with 10 ml ice-cold PBS per plate and remove PBS completely.</li>
 <li>Place plates on a tray with ice and irradiate uncovered with 0.15 J/cm2 of 365 nm UV light in a Stratalinker 2400 (Stratagene) or similar device.</li>
 <li>Scrape cells off with a rubber policeman in 1 ml PBS per plate, transfer to 50 ml centrifugation tubes and collect by centrifugation at 500 x g for 5 min at 4&deg;C and discard the supernatant. 100 x 106 HEK293 cells (10 15 cm plates) will yield approx. 1 ml of wet cell pellet.</li>
 <li>(optional) Unless you want to continue directly with cell lysis, shock freeze the cell pellet in liquid nitrogen and store at -80&deg;C. Cell pellets can be stored for at least 12 months.</li>
</ol>
Cell lysis and RNaseT1 digest
<ol>
	<li>Take up cell pellet of crosslinked cells in 3 volumes of 1x NP40 lysis buffer and incubate on ice for 10 min.</li>
 <li>Clear cell lysate by centrifugation at 13,000 x g for 15 min at 4&deg;C.</li>
 <li>Clear the lysate further by filtering it through a 0.2 &mu;m membrane syringe filter (Pall Acrodisc or equivalent).</li>
 <li>Add RNase T1 (Fermentas, 10,000 U/&mu;l) to a final concentration of 1 U/&mu;l and incubate in a water bath for 15 min at 22&deg;C. Cool reaction subsequently for 5 min on ice before proceeding.</li>
</ol>
Immunoprecipitation and recovery of crosslinked target RNA fragments
Using the magnetic separator
Follow these guidelines throughout the sample preparation to prevent the magnetic beads from drying out.
<ol>
	<li>Place the tube containing the beads on the magnetic stand for 1 2 minutes.</li>
 <li>Add the buffer to the tube while the tube is on the magnetic separator.</li>
 <li>Cap the tube, remove it from the magnetic separator, and resuspend the beads. You can resuspend the beads by flicking the tube with your finger or use a vortexer set at 5 6.</li>
 <li>Centrifuge briefly to collect any beads that may remain in the tube cap.</li>
 <li>Repeat steps 1 through 4 as required.</li>
</ol>
Preparation of magnetic beads
<ol>
	<li>Transfer 10 &mu;l of Dynabeads Protein G magnetic particles (Invitrogen) per ml cell lysate (for a typical experiment it should be approx. 40 50 &mu;l of beads) to a 1.5 ml microfuge tube. Wash beads twice with 1 ml of citrate-phosphate buffer.</li>
 <li>Resuspend in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.</li>
 <li>Add 0.25 &mu;g of anti-FLAG M2 monoclonal antibody (Sigma) per ml suspension and incubate on a rotating wheel for 40 min at room temperature.</li>
 <li>Wash beads twice in 1 ml of citrate-phosphate buffer to remove unbound antibody.</li>
 <li>Resuspend beads in twice the volume of citrate-phosphate buffer relative to the original volume of bead suspension.</li>
</ol>
Immunoprecipitation (IP), second RNase T1 digestion, and dephosphorylation
<ol>
	<li>Add 20 &mu;l of freshly prepared antibody-conjugated magnetic beads per ml of partial RNase T1 treated cell lysate and incubate in 15 ml centrifugation tubes on a rotating wheel for 1 h at 4&deg;C.</li>
 <li>Collect magnetic beads on a magnetic particle collector for 15 and 50 ml centrifugation tubes (Invitrogen) and transfer to 1.5 ml microfuge tubes.</li>
 <li>Wash beads 3 times in 1 ml of IP wash buffer.</li>
 <li>Add RNaseT1 (Fermentas, 10,000 U/&mu;l) to a final concentration of 100 U/&mu;l and incubate the bead suspension in a water bath for 15 min at 22 &deg;C. Cool subsequently on ice for 5 min.</li>
 <li>Wash beads 3 times in 1 ml of high-salt wash buffer. </li>
 <li>Resuspend beads in 1 volume of dephosphorylation buffer</li>
 <li>Add calf intestinal alkaline phosphatase (CIAP, NEB) to a final concentration of 0.5 U/&mu;l, and incubate the suspension for 10 min at 37&deg;C.</li>
 <li>Wash beads twice in 1 ml of phosphatase wash buffer</li>
 <li>Wash beads twice in polynucleotide kinase (PNK) buffer without DTT (the DTT concentration necessary for the enzymatic reaction is high enough to damage the magnetic beads).</li>
 <li>Resuspend beads in one original bead volume of PNK buffer</li>
</ol>
Radiolabeling of RNA segments crosslinked to immunoprecipitated proteins
<ol>
 <li>To the bead suspension described above, add &Upsilon;-32P-ATP to a final concentration of 0.5 &mu;Ci/&mu;l and T4 PNK (NEB) to 1 U/&mu;l in one original bead volume. Incubate the suspension for 30 min at 37&deg;C.</li>
 <li>Add non-radioactive ATP to obtain a final concentration of 100 &mu;M and incubate for another 5 min at 37&deg;C.</li>
 <li>Wash the magnetic beads 5 times with 800 &mu;l of PNK buffer without DTT.</li>
 <li>Resuspend the beads in 70 &mu;l of SDS-PAGE loading buffer.</li>
</ol>
SDS-PAGE and electroelution of crosslinked RNA-protein complexes from gel slices
<ol>
	<li>Incubate the radiolabeled suspension for 5 min in a heat block at 95&deg;C to denature and release the immunoprecipitated RBP with crosslinked RNA and vortex.</li>
 <li>Remove the magnetic beads on the separator and transfer the supernatant to a clean 1.5 ml microfuge tube.</li>
 <li>Load 40 &mu;l of the supernatant per well of a Novex Bis-Tris 4-12% (Invitrogen) precast polyacrylamide gel and run the gel for 55 min at 200 V.</li>
 <li>Disassemble the gel chamber and dismantle the gel, leaving it mounted on one plate. To facilitate the alignment of the gel to the phosphorimager paper printout, we recommend implanting three tiny radioactive gel pieces asymmetrically at three of the four corners of the gel. Radioactive gel pieces can be collected from gels that were previously used to purify radiolabeled synthetic oligonucleotides. Wrap the gel in plastic film (e.g. Saran wrap) to avoid contamination.</li>
 <li>Expose the gel to a blanked phosphorimager screen for 1 h and visualize on a phosphorimager.</li>
 <li>Align the gel on top of the phosphorimager printout using the implanted gel pieces for orientation. Cut out the bands that correspond to the expected size of RBP (IGF2BP1, approx. 75 kDa) and transfer to a D-Tube Dialyzer Midi Tube and add 800 &mu;l 1x SDS running buffer.</li>
 <li>Electroelute the crosslinked RNA-RBP complex in 1x SDS running buffer at 100 V for 2 h. excised from the gel and electroeluted in a D-Tube Dialyzer Midi (Novagen) in 800 &mu;l SDS running buffer according to the instructions of the manufacturer.</li>
</ol>
Proteinase K digestion
<ol>
 <li>Add an equal volume of 2x Proteinase K Buffer with respect to the electroeluate, followed by the addition of Proteinase K (Roche) to a final concentration of 1.2 mg/ml. Incubate for 30 min at 55&deg;C.</li>
 <li>Recover the RNA by acidic phenol/chloroform/IAA extraction (25:24:1, pH 4.0) followed by a chloroform extraction. Add 1 &mu;l of glycogen (10 mg/ml stock) precipitate the RNA by adding 3 volumes of ethanol. Dissolve the pellet in 10.5 &mu;l water.</li>
</ol>
cDNA library preparation and deep sequencing
Carry the recovered RNA through a standard cDNA library preparation protocol originally described for cloning of small regulatory RNAs 19. The first step, 3' adapter ligation, was carried out as described on a 20 &mu;l scale using 10.5 &mu;l of the recovered RNA. Use the Solexa sequencing adapter sets described. Depending on the amount of RNA recovered, 5'-adapter-3'-adapter products without inserts may be detected after amplification of the cDNA as additional PCR bands. In such cases, excise the longer PCR product of expected size from a 3% NuSieve low-melting point agarose gel, elute the PCR product from the gel pieces using the GelElute kit (Qiagen) and sequence using the Solexa technology. One Solexa sequencing run usually affords between 6 and 10 million sequence reads that are enough for a transcriptome wide coverage of the binding sites of RNA binding proteins.
Bioinformatic analysis
Careful bioinformatic analysis of the sequence reads needs to be done to obtain meaningful insights into the RNA binding sites for the examined RBP, such as the RNA recognition element, the preferred binding regions the RBP has (exonic vs. intronic, coding sequence vs. untranslated sequence). The sequence reads need to be aligned against the genome and EST databases. We usually use reads mapping uniquely to the genome with up to one mismatch, insertion or deletion to build clusters of sequence reads that can then be further analyzed. The frequency of characteristic mutations in the clustered sequenced reads, T to C transitions when using 4-SU and G to A transitions when using 6-SG, are indicative of successfully crosslinked sequences. In our experience uncrosslinked RNAs labeled with 4-SU show a background mutation rate of approximately 20%. This rate is increases to approx. 50-80% upon crosslinking.
A detailed description of the bioinformatic analysis can be found in the Supplementary material of the publication by Hafner et al.18
Optional Steps
Determination of incorporation levels of 4-thiouridine into total RNA
Isolate total RNA from the cell line stably expressing the RBP of interest after growing in medium supplemented with 100 &mu;M 4SU 16 h prior to harvest. As a control, harvest cells grown without 4SU addition. Isolate total RNA by addition of 3 volumes of Trizol reagent (Sigma) to the washed cell pellets following the manufacturer s instructions. was Further purify total RNA using Qiagen RNeasy according to the manufacturer's protocol. To prevent oxidization of 4SU during RNA isolation and analysis, add 0.1 mM dithiothreitol (DTT) to the wash buffers and subsequent enzymatic steps. Digest and dephosphorylated total RNA to single nucleosides for HPLC analysis as described before 20. Briefly, in a 30 &mu;l volume, incubate 40 &mu;g of purified total RNA were for 16 h at 37&deg;C with 0.4 U bacterial alkaline phosphatase (Worthington Biochemical) and 0.09 U snake venom phosphodiesterase (Worthington Biochemical). As a reference standard, use a synthetic 4SU-labeled RNA, (we standardly use CGUACGCGGAAUACUUCGA(4SU)U) and also subject it to complete enzymatic digestion. Separate the resulting mixtures of ribonucleosides by HPLC on a Supelco Discovery C18 (bonded phase silica 5 &mu;M particle, 250 x 4.6 mm) reverse phase column (Bellefonte PA, USA). HPLC buffers are 0.1 M TEAA in 3% acetonitrile (A) and 90% acetonitrile in water (B). Use an isocratic gradient: 0% B for 15 min, 0 to 10 % B for 20 min, 10 to 100% B for 30 min. Apply a 5 min 100 % B wash applied between runs to clean the HPLC column.
Representative Results
<img src="/files/ftp_upload/2034/2034fig1.jpg" alt="Figure 1" /> Figure 1 (right panel) shows a representative result of a PAR-CliP performed with cell lines expressing FLAG/HA-tagged IGF2BP1 with 4-SU and 6-SG. Note that the crosslinking efficiency of 6-SG for IGF2BP1 is lower than the crosslinking efficiency for 4-SU. The lower crosslinking efficiency will result in a higher background of sequences derived from fragments of abundant cellular RNAs and therefore you should consider scaling up the experiment when using less efficient photoreactive nucleosides. 
The left panel of Figure 1 shows a comparison of using different photoreactive uridine analogs that could be potentially used for PAR-CliP compared to traditional UV 254 nm crosslinking. 
The intensity of the radioactive band of the correct length gives you a good idea whether the PAR-CliP experiment has worked and you have isolated sufficient RNA to carry through a small RNA sequencing protocol (step-by-step description for cDNA library preparation of small RNAs sequencing can be found in 19). The frequency of characteristic mutations in the sequenced reads, T to C transitions when using 4-SU and G to A transitions when using 6-SG, are indicative of successfully crosslinked sequences. In our experience uncrosslinked RNAs labeled with 4-SU show a background mutation rate of approximately 20%. This rate is increases to approx. 50-80% upon crosslinking.

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Biochem. 112 (2), 323-323 (1980).</li><li>Mayrand, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+nuclear+ribonucleoprotein:+identification+of+proteins+in+contact+with+poly(A)++heterogeneous+nuclear+RNA+in+living+HeLa+cells.">Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.</a> The Journal of Cell Biology. 90 (2), 380-380 (1981).</li><li>Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+heterogeneous+nuclear+RNA-protein+complexes+in+vivo+with+monoclonal+antibodies.">Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.</a> Mol. Cell. Biol. 4 (6), 1104-11 (1984).</li><li>Adam, S. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+discovery+of+endogenous+Argonaute+binding+sites+in+Caenorhabditis+elegans.">Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.</a> Nat. Struct. Mol. Biol. , (2010).</li><li>Hafner, M. <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. , (2010).</li><li>Hafner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+microRNAs+and+other+small+regulatory+RNAs+using+cDNA+library+sequencing.">Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.</a> Methods. 44 (1), 3-3 (2008).</li><li>Andrus, A., Kuimelis, R. 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T.T. is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.

Buffers and reagents
Growth medium HEK293 cells
<ul>
	<li>DMEM</li>
	<li>10% FBS</li>
	<li>2 mM L-Glutamine</li>
	<li>100 U/ml penicillin</li>
	<li>100 U/ml streptomycin</li>
	<li>100 &mu;g/ml hygromycin</li>
	<li>15 &mu;g/ml blasticidin</li>
</ul>
4-thiouridine stock solution (1 M)
<ul>
	<li>260.27 mg 4-thiouridine</li>
	<li>1 ml DMSO</li>
</ul>
Doxycyclin stock (10 mg/ml)
<ul>
	<li>10 mg doxycyclin</li>
	<li>1 ml DMSO</li>
</ul>
1x NP40 lysis buffer
Prepare a stock of 5x buffer without DTT and protease inhibitors. Add DTT and protease inhibitor directly before the experiment.
<ul>
	<li>50 mM HEPES, pH 7.5</li>
	<li>150 mM KCl</li>
	<li>2 mM EDTA</li>
	<li>1 mM NaF</li>
	<li>0.5% (v/v) NP40</li>
	<li>0.5 mM DTT</li>
	<li>complete EDTA-free protease inhibitor cocktail (Roche)</li>
</ul>
Citrate-Phosphate buffer
<ul>
	<li>4.7 g/l citric acid</li>
	<li>9.2 g/l Na2HPO4</li>
	<li>pH 5.0</li>
</ul>
IP-wash buffer
<ul>
	<li>50 mM HEPES-KOH, pH 7.5</li>
	<li>300 mM KCl</li>
	<li>0.05% (v/v) NP40</li>
	<li>0.5 mM DTT (add directly before experiment)</li>
	<li>complete EDTA-free protease inhibitor cocktail (Roche) (add directly before experiment)</li>
</ul>
High-salt wash buffer
<ul>
	<li>50 mM HEPES-KOH, pH 7.5</li>
	<li>500 mM KCl</li>
	<li>0.05% (v/v) NP40</li>
	<li>0.5 mM DTT (add directly before the experiment)</li>
	<li>complete EDTA-free protease inhibitor cocktail (Roche) (add directly before the experiment)</li>
</ul>
Dephosphorylation buffer
<ul>
	<li>50 mM Tris-HCl, pH 7.9</li>
	<li>100 mM NaCl</li>
	<li>10 mM MgCl2</li>
	<li>1 mM DTT</li>
</ul>
Phosphatase wash buffer
<ul>
	<li>50 mM Tris-HCl, pH 7.5</li>
	<li>20 mM EGTA</li>
	<li>0.5% (v/v) NP40</li>
</ul>
Polynucleotide kinase (PNK) buffer without DTT
<ul>
	<li>50 mM Tris-HCl, pH 7.5</li>
	<li>50 mM NaCl</li>
	<li>10 mM MgCl2</li>
</ul>
PNK buffer
<ul>
	<li>50 mM Tris-HCl, pH 7.5</li>
	<li>50 mM NaCl</li>
	<li>10 mM MgCl2</li>
	<li>5 mM DTT</li>
</ul>
SDS PAGE loading buffer
<ul>
	<li>10% glycerol (v/v)</li>
	<li>50 mM Tris-HCl, pH 6.8</li>
	<li>2 mM EDTA</li>
	<li>2% SDS (w/v)</li>
	<li>100 mM DTT</li>
	<li>0.1% bromophenol blue</li>
</ul>
2x Proteinase K buffer
<ul>
	<li>100 mM Tris-HCl, pH 7.5</li>
	<li>150 mM NaCl</li>
	<li>12.5 mM EDTA</li>
	<li>2% (w/v) SDS</li>
</ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

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

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.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...