Name: single-step purification of macromolecular complexes using rna attached to biotin and a photo-cleavable linker
Uploaded: 2019-01-03T13:00:00
Description: Watch this Scientific Journal Video about Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker at JoVE.com

We thank our colleagues and collaborators for their contribution to our work. This study was supported by the NIH grant GM 29832.

1. Substrate Preparation
NOTE: RNA substrates shorter than ~65 nucleotides can be synthesized chemically with biotin (B) and the photo-cleavable (pc) linker (together referred to as photo-cleavable biotin or pcB) covalently attached to the RNA 5&#8217; end (cis configuration). RNA substrates containing significantly longer binding sites need to be generated in vitro by T7 (or SP6) transcription and subsequently annealed to a short complementary adaptor oligonucleotide containin...

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F., Dominski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+composition+of+catalytically+active+U7-dependent+processing+complexes+assembled+on+histone+pre-mRNA+containing+biotin+and+a+photo-cleavable+linker.">Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker.</a> Nucleic Acids Research. 46 (9), 4752-4770 (2018).</li><li>Dominski, Z., Yang, X. C., Kaygun, H., Dadlez, M., Marzluff, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3+'+exonuclease+that+specifically+interacts+with+the+3+'+end+of+histone+mRNA.">A 3 ' exonuclease that specifically interacts with the 3 ' end of histone mRNA.</a> Molecular Cell. 12 (2), 295-305 (2003).</li><li>Yang, X. C., Purdy, M., Marzluff, W. F., Dominski, Z. <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+3'hExo,+a+3'+exonuclease+specifically+interacting+with+the+3'+end+of+histone+mRNA.">Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA.</a> Journal of Biological Chemistry. 281 (41), 30447-30454 (2006).</li><li>Tan, D., Marzluff, W. F., Dominski, Z., Tong, L. <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+histone+mRNA+stem-loop,+human+stem-loop+binding+protein,+and+3'hExo+ternary+complex.">Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3'hExo ternary complex.</a> Science. 339 (6117), 318-321 (2013).</li><li>Mayeda, A., Krainer, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+HeLa+cell+nuclear+and+cytosolic+S100+extracts+for+in+vitro+splicing.">Preparation of HeLa cell nuclear and cytosolic S100 extracts for in vitro splicing.</a> Methods in Molecular Biology. 118, 309-314 (1999).</li><li>Dominski, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'+end+processing+of+Drosophila+histone+pre-mRNAs:+Requirement+for+phosphorylated+dSLBP+and+co-evolution+of+the+histone+pre-mRNA+processing+system.">3' end processing of Drosophila histone pre-mRNAs: Requirement for phosphorylated dSLBP and co-evolution of the histone pre-mRNA processing system.</a> Molecular and Cellular Biology. 22, 6648-6660 (2002).</li><li>Dominski, Z., Yan, X. C., Purdy, M., Marzluff, W. 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The method described here is straightforward and besides incorporating a photo-cleavable linker and the UV-elution step does not differ from the commonly used methods that take advantage of the extremely strong interaction between biotin and streptavidin. The UV-elution step is very efficient, typically releasing more than 75% of the immobilized RNA and associated proteins from streptavidin beads, leaving behind a high background of proteins that non-specifically bind to the beads. By eliminating this background, the UV-...

In eukaryotes, RNA polymerase II-generated mRNA precursors (pre-mRNAs) undergo several maturation events in the nucleus before becoming fully functional mRNA templates for protein synthesis in the cytoplasm. One of these events is 3&#39; end processing. For the vast majority of pre-mRNAs, 3&#39; end processing involves cleavage coupled to polyadenylation. This two-step reaction is catalyzed by a relatively abundant complex consisting of more than 15 proteins1. Animal replication-dependent histone pre-mRNAs are processed at the 3&#39; end by a different mechanism in which the key role is played by U7 snRNP, a low abundance complex consisting of U7 snRNA of ~60 nucleotides and multiple proteins2,3. The U7 snRNA base pairs with a specific sequence in histone pre-mRNA and one of the subunits of the U7 snRNP catalyzes the cleavage reaction, generating mature histone mRNA without a poly(A) tail. 3&#39; end processing of histone pre-mRNA also requires Stem-Loop Binding Protein (SLBP), which binds a conserved stem-loop located upstream of the cleavage site and enhances the recruitment of the U7 snRNP to the substrate2,3. Studies aimed at identifying individual components of the U7 snRNP have been challenging due to the low concentration of the U7 snRNP in animal cells and the tendency of the complex to dissociate or undergo partial proteolysis during purification as a result of using mild detergents4,5,6, high salt washes and/or multiple chromatographic steps7,8,9.
Recently, to determine the composition of the U7-dependent processing machinery, a short fragment of histone pre-mRNA containing biotin at either 3&#39; or 5&#39; was incubated with a nuclear extract and the assembled complexes were captured on streptavidin-coated agarose beads5,6,10. Due to the exceptionally strong interaction between biotin and streptavidin, proteins immobilized on streptavidin beads were eluted under denaturing conditions by boiling in SDS and analyzed by silver staining and mass spectrometry. While this simple approach identified a number of components of the U7 snRNP, it yielded relatively crude samples, often contaminated with a large number of background proteins nonspecifically bound to streptavidin beads, potentially masking some components of the processing machinery and preventing their detection on silver stained gels5,6,10. Importantly, this approach also precluded any functional studies with the isolated material and its further purification to homogeneity by additional methods.
A number of modifications were proposed over time to address the virtually irreversible nature of the biotin/streptavidin interaction, with most of them being designed to either weaken the interaction or to provide a chemically cleavable spacer arm in the biotin-containing reagents11,12. The downside of all these modifications was that they significantly reduce the efficiency of the method and/or often required non-physiological conditions during the elution step, jeopardizing either the integrity or activity of the purified proteins.
Here, we describe a different approach to resolve the inherent problem of the biotin/streptavidin strategy by using RNA substrates in which biotin is covalently attached to the 5&#39; end via a photo-cleavable 1-(2-nitrophenyl)ethyl moiety that is sensitive to long wave UV13,14. We tested this approach for the purification of the limiting U7-dependent processing machinery from Drosophila and mammalian nuclear extracts15. Following a short incubation of histone pre-mRNA containing biotin and the photo-cleavable linker with a nuclear extract, the assembled processing complexes are immobilized on streptavidin beads, thoroughly washed and gently released to solution in a native form by exposure to ~360 nm UV light. The UV-elution method is very efficient, fast and straightforward, yielding sufficient amounts of the U7 snRNP to visualize its components by sliver staining from&#160;as little as 100 &#181;L of the extract15. The UV-eluted material is free of background proteins and suitable for direct mass spectrometry analysis, additional purification steps and enzymatic assays. The same method may be adopted for the purification of other RNA/protein complexes that require relatively short RNA binding sites. Biotin and the photo-cleavable linker can also be covalently attached to single- and double-stranded DNA, potentially extending the UV-elution method for the purification of various DNA/protein complexes.
Chemical synthesis of RNA substrates containing covalently attached biotin and the photo-cleavable linker is practical only with the sequences that do not exceed ~65 nucleotides, becoming expensive and inefficient for significantly longer sequences. To address this problem, we also developed an alternative approach that is suitable for much longer RNA binding targets. In this approach, RNA of any length and nucleotide sequence is generated in vitro by T7 or SP6 transcription and annealed to a short complementary oligonucleotide that contains biotin and the photo-cleavable linker at the 5&#39; end (trans configuration). The resultant duplex is subsequently used to purify individual binding proteins or macromolecular complexes on streptavidin beads following the same protocol described for the RNA substrates containing photo-cleavable biotin attached covalently (cis configuration). With this modification, the photo-cleavable biotin can be used in conjunction with in vitro generated transcripts containing hundreds of nucleotides, extending the UV-elution method for the purification of a broad range of RNA/protein.

<table>
	<tbody>
		<tr>
			<td>Streptavidin-Agarose</td>
			<td>Sigma</td>
			<td>S1638-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>High-intensity, long-wave UV lamp</td>
			<td>Cole-Parmer</td>
			<td>UX-97600-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Replacement bulb</td>
			<td>Cole-Parmer</td>
			<td>UX-97600-19</td>
			<td>100 Watts Mercury H44GS-100M bulb emitting 366 nm UV light (Sylvania)</td>
		</tr>
		<tr>
			<td>RNAs and oligonucleotides containing biotin and photo-cleavable linker in cis</td>
			<td>Dharmacon (Lafayette, CO) or Integrated DNA Technologies, Inc. (Coralville, IA)</td>
			<td></td>
			<td>Requst a quote in Dharmacon</td>
		</tr>
		<tr>
			<td>Beckman GH 3.8 swing bucket rotor</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RiboMAX Large Scale RNA Production Systems (T7 polymerase)</td>
			<td>Promega</td>
			<td>P1300</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboMAX Large Scale RNA Production Systems (SP6 polymerase)</td>
			<td>Promega</td>
			<td>P1280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Silver Stain Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>24612</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-step purification of macromolecular complexes using rna attached to biotin and a photo-cleavable linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

The UV-elution method was tested with two chemically synthesized RNA substrates covalently attached at the 5&#39; end to the pcB moiety (cis configuration): pcB-SL (Figure 1) and pcB-dH3/5m RNAs (Figure 2). The 31-nucleotide pcB-SL RNA contains a stem-loop structure followed by a 5-nucleotide single stranded tail and its sequence is identical to the 3&#39; end of mature histone mRNA (i.e., after the cleavage of ...

Watch this Scientific Journal Video about Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker at JoVE.com

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

This method should be of interest to researchers who are trying to isolate RNA protein complexes and identify their composition by mass spectrometry. We believe our protocol will be especially useful for purifying complexes that exist in cells in limited amounts and tend to dissociate during long multi-step purification procedures. The main advantage of this method is that it involves only a single purification step and could be used with RNA binding sites of any length and sequence.Demonstrating some of the steps will be Xiao-cui Yang, who is a senior research technician in our group. For binding sites significantly longer than 65 nucleotides, obtain T7 generated RNA and an adaptor PCB oligonucleotide as outlined in the text protocol. Mix 20 picomoles of the RNA with 100 picomoles of the adaptor oligonucleotide in a 1.5 milliliter tube containing 100 microliters of binding buffer.Place the tube in boiling water and let it incubate for 5 minutes. Then allow the water to cool down to room temperature. First supplement 1 milliliter of mouse nuclear extract with 80 millimolar EDTA at pH 8 to a final concentration of 10 millimolar.Then add between 5 and 10 picomoles of the RNA substrate that has been tagged with the PCB moiety. Incubate on ice for 5 minutes, making sure to occasionally mix the sample. Next use a pre-cooled microcentrifuge at 4 degrees Celsius to centrifuge the mixture at 10, 000 times g for 10 minutes to remove any potential precipitates.Carefully collect the supernatant into a new tube on ice, while being careful to avoid transferring the pellet. Transfer approximately 100 microliters of streptavidin agarose bead suspension to a 1.5 milliliter tube. Add approximately 1 milliliter of binding buffer to begin washing the beads.Centrifuge the tube at 25 times g for 2 to 3 minutes and aspirate the supernatant. Repeat this washing and centrifugation process 2 to 3 times to equilibrate the beads with the binding buffer. Load the previously collected supernatant, which contains the assembled complex over the equilibrated beads.Using a tube rotator, rotate the tube for 1 hour at 4 degrees Celsius to immobilize the RNA and bound complexes onto the beads. Next spin the tube at 25 times g for 2 to 3 minutes to collect the beads at the bottom of the tube. Aspirate the supernatant.And rinse the beads twice with binding buffer, using the previously described washing process. After removing the supernatant of the second wash, add 1 milliliter of binding buffer and rotate the sample for 1 hour at 4 degrees Celsius. Spin down the sample at 25 times g for 2 to 3 minutes.Next add 1 milliliter of binding buffer and transfer the suspension to a new tube. Rotate the sample at 4 degrees Celsius for up to 1 hour. Centrifuge the immobilized complexes at 25 times g for 2 to 3 minutes.Then aspirate supernatant, and add 200 microliters of binding buffer. Transfer the beads to a 500 microliter tube. Turn on a high intensity UV lamp that emits UV light at 365 nanometers and allow it to reach full brilliance.Fill up the bottom of a Petri dish with tightly packed ice stacking it over the dish's lid. Briefly vortex the tube containing the immobilized complexes. Then place it horizontally on the ice and cover it with the pre-warmed lamp, making sure that the sample is 2 to 3 centimeters from the surface of the bulb.Irradiate the sample for a total of 30 minutes while frequently inverting and vortexing the tube to ensure uniform exposure and to prevent overheating. After this, spin down the beads at 25 times g for 2 to 3 minutes and collect the supernatant. Centrifuge this supernatant once more using the same conditions.Collect the resulting supernatant making sure to leave a small amount at the bottom of the tube to avoid transferring any residual beads. Using SDS-PAGE electrophoresis, separate a fraction of the UV eluted supernatant and the corresponding fraction from the material left on the beads after UV elution. Next use a commercially available silver staining kit to stain the gel.Evaluate the efficiency of UV elution by comparing the intensities of the proteins present in the UV eluted supernatant and those left on the beads following UV irradiation. Excise the protein bands of interest and determine their identities by mass spectrometry. After this, directly analyze a fraction of the UV eluted supernatant by mass spectrometry to determine the entire proteome of the purified material in an unbiased manner.In this study, stem loop RNA tagged with photo-cleavable biotin is incubated with 1 milliliter of a cytoplasmic S100 extract obtained from mice myeloma cells. And the effect and efficiency of UV elution is analyzed by silver staining. A number of proteins are detected on the streptavidin beads before UV elution.Irradiation with long wave UV releases only some of these proteins into the supernatant leaving a non-specific background on the beads. This emphasizes the importance of the UV elution step. Mass spectrometry identifies the major UV eluted proteins as 3'hExo and SLBP with SLBP being represented by both the full length protein and a number of shorter degradation products.Smaller amounts of other proteins identified as various RNA binding proteins also selectively released into solution by the UV irradiation. UV elution of immobilized Drosophila histone pre-mRNA tagged with photo-cleavable biotin incubated with a nuclear extract resulted in a selective release of only a small number of proteins into the supernatant with an intense background of non-specific proteins remaining on the beads. Mass spectrometry identifies these proteins to be components of the U7 snRNP.They are not detected in the presence of processing competitors that block binding of U7 snRNP to histone pre-mRNA. It is important to use high intensity UV lamp and place it a close distance to the irradiated sample while also making sure that it doesn't overheat. The UV elution method yields native RNA protein complexes that lack major contaminants and are directly suitable for additional purification steps and functional assays.Avoid eye and skin exposure during UV irradiation.

single-step-purification-macromolecular-complexes-using-rna-attached

Research

JoVE Journal

Biochemistry

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.