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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C., 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=A+Complex+Containing+the+CPSF73+Endonuclease+and+Other+Polyadenylation+Factors+Associates+with+U7+snRNP+and+Is+Recruited+to+Histone+Pre-mRNA+for+3+'-End+Processing.">A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3 '-End Processing.</a> Molecular and Cellular Biology. 33 (1), 28-37 (2013).</li><li>Shimkus, M., Levy, J., Herman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemically+cleavable+biotinylated+nucleotide:+usefulness+in+the+recovery+of+protein-DNA+complexes+from+avidin+affinity+columns.">A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (9), 2593-2597 (1985).</li><li>Hirsch, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocleavable+biotin+phosphoramidite+for+5'-end-labeling,+affinity+purification+and+phosphorylation+of+synthetic+oligonucleotides.">Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.</a> Nucleic Acids Research. 24 (2), 361-366 (1996).</li><li>Olejnik, J., Krzymanska-Olejnik, E., Rothschild, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocleavable+affinity+tags+for+isolation+and+detection+of+biomolecules.">Photocleavable affinity tags for isolation and detection of biomolecules.</a> Methods in Enzymology. 291, 135-154 (1998).</li><li>Skrajna, A., Yang, X. C., Dadlez, M., Marzluff, W. 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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=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.

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JoVE Journal

Biochemistry

7.1K visualizações.  University of North Carolina at Chapel Hill. Esse método deve ser de interesse para pesquisadores que estão tentando isolar complexos proteicos de RNA e identificar sua composição por espectrometria de massa. Acreditamos que nosso protocolo será especialmente útil para purificar complexos que existem em células em quantidades limitadas e tendem a dissociar durante longos procedimentos de purificação em várias etapas. A principal vantagem deste método é que ele envolve apenas uma única etapa de purificação e poderia ser us...