Name: global identification of co-translational interaction networks by selective ribosome profiling
Uploaded: 2021-10-07T13:45:00
Description: Watch this Scientific Journal Video about Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling at JoVE.com

We would like to thank all the lab members for fruitful discussions and Muhammad Makhzumy for the critical reading of the manuscript. This work was funded by ISF (Israeli Science Foundation) grant 2106/20.

1. Generating strains for Selective Ribosome Profiling
NOTE: Selective Ribosome Profiling (SeRP) is a method that relies on affinity purification of factors of interest, to assess their mode of interaction with ribosomes-nascent chain complexes. Homologous recombination9, as well as CRISPR/Cas910 based methods are utilized to fuse various factors of interest with tags for affinity purifications. Such tags are GFP, for GFP-trap ...

<ol>
	<li>Oh, E., 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=Selective+ribosome+profiling+reveals+the+cotranslational+chaperone+action+of+trigger+factor+in+vivo.">Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.</a> Cell. 147 (6), 1295-1308 (2011).</li><li>Shiber, A., 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=Cotranslational+assembly+of+protein+complexes+in+eukaryotes+revealed+by+ribosome+profiling.">Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling.</a> Nature. 561 (7722), 268-272 (2018).</li><li>Becker, A. H., Oh, E., Weissman, J. S., Kramer, G., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+ribosome+profiling+as+a+tool+for+studying+the+interaction+of+chaperones+and+targeting+factors+with+nascent+polypeptide+chains+and+ribosomes.">Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes.</a> Nature Protocols. 8 (11), 2212-2239 (2013).</li><li>Galmozzi, C. V., Merker, D., Friedrich, U. A., D&#246;ring, K., Kramer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+ribosome+profiling+to+study+interactions+of+translating+ribosomes+in+yeast.">Selective ribosome profiling to study interactions of translating ribosomes in yeast.</a> Nature Protocols. , (2019).</li><li>Knorr, A. G., 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=Ribosome-NatA+architecture+reveals+that+rRNA+expansion+segments+coordinate+N-terminal+acetylation.">Ribosome-NatA architecture reveals that rRNA expansion segments coordinate N-terminal acetylation.</a> Nature Structural and Molecular Biology. 26 (1), 35-39 (2019).</li><li>Matsuo, Y., Inada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+collision+sensor+Hel2+functions+as+preventive+quality+control+in+the+secretory+pathway.">The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway.</a> Cell Reports. 34 (12), (2021).</li><li>D&#246;ring, K., 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=Profiling+Ssb-Nascent+chain+interactions+reveals+principles+of+Hsp70-assisted+folding.">Profiling Ssb-Nascent chain interactions reveals principles of Hsp70-assisted folding.</a> Cell. , (2017).</li><li>Chartron, J. W., Hunt, K. C. L., Frydman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+signal-independent+SRP+preloading+during+membrane+targeting.">Cotranslational signal-independent SRP preloading during membrane targeting.</a> Nature. 536 (7615), 224-228 (2016).</li><li>Janke, 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+versatile+toolbox+for+PCR-based+tagging+of+yeast+genes:+New+fluorescent+proteins,+more+markers+and+promoter+substitution+cassettes.">A versatile toolbox for PCR-based tagging of yeast genes: New fluorescent proteins, more markers and promoter substitution cassettes.</a> Yeast. 21 (11), 947-962 (2004).</li><li>Levi, O., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+CRISPR/Cas9+Toolbox+for+Gene+Engineering+in+S.+cerevisiae.">Expanding the CRISPR/Cas9 Toolbox for Gene Engineering in S. cerevisiae.</a> Current Microbiology. 77 (3), 468-478 (2020).</li><li>Giannoukos, G., 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=Efficient+and+robust+RNA-seq+process+for+cultured+bacteria+and+complex+community+transcriptomes.">Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes.</a> Genome Biology. 13 (3), 23 (2012).</li><li>. Illumina Index Adapters - Pooling Guide Available from: <a target="_blank" href="https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/index-adapters-pooling-guide-1000000041074-05.pdf">https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/index-adapters-pooling-guide-1000000041074-05.pdf</a> (2019)</li><li>Kanagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bias+and+artifacts+in+multitemplate+polymerase+chain+reactions+(PCR).">Bias and artifacts in multitemplate polymerase chain reactions (PCR).</a> Journal of Bioscience and Bioengineering. 96 (4), 317-323 (2003).</li><li>Bertolini, M., 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=Interactions+between+nascent+proteins+translated+by+adjacent+ribosomes+drive+homomer+assembly.">Interactions between nascent proteins translated by adjacent ribosomes drive homomer assembly.</a> Science. 371 (6524), (2021).</li><li>Kramer, G., Shiber, A., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cotranslational+maturation+of+newly+synthesized+proteins.">Mechanisms of cotranslational maturation of newly synthesized proteins.</a> Annual Review of Biochemistry. 88, 337-364 (2019).</li><li>Joazeiro, C. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+ribosome-associated+protein+quality+control.">Mechanisms and functions of ribosome-associated protein quality control.</a> Nature Reviews Molecular Cell Biology. 20 (6), 368-383 (2019).</li><li>Beaupere, C., Chen, R. B., Pelosi, W., Labunskyy, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+quantification+of+translation+in+budding+yeast+by+ribosome+profiling.">Genome-wide quantification of translation in budding yeast by ribosome profiling.</a> Journal of Visualized Experiments: JoVE. (130), e56820 (2017).</li></ol>

Here, the protocol details the Selective Ribosome Profiling approach for capturing co-translational interactions in near codon resolution.&#160;As the ribosome rises as a hub for coordinating the nascent-chain emergence into the crowded cytoplasm, this is a crucial method to identify and characterize the various co-translational interactions required to ensure a functional proteome, as well as for studying various diseases. To date, SeRP is the only method that can capture and characterize these interactions, in a direct...

Selective Ribosome Profiling (SeRP) is the only method, to date, that captures and characterizes co-translational interactions, in vivo, in a direct manner1,2,3,4,5,6. SeRP enables global profiling of interactions of any factor with translating ribosomes in&#160;near codon resolution2,7.
The method relies on flash freezing of growing cells and preserving active translation. Cell lysates are then treated with RNase I to digest all mRNA in the cell except ribosome-protected mRNA fragments termed &#34;ribosome footprints&#34;.&#160;The sample is then split into two parts; one part is directly used for the isolation of all the cellular ribosomal footprints, representing all ongoing translation in the cell. The second part is used for the affinity-purification of the specific subset of ribosomes associated with a factor of interest, for example: modifying enzymes, translocation factors, folding chaperones, and complex-assembly interactions. The affinity-purified ribosomal footprints are collectively termed the interactome. Then, the ribosome-protected mRNAs are extracted and used for cDNA library generation, followed by deep sequencing.
Comparative analysis of the total translatome and interactome samples allows for the identification of all orfs which associate with the factor of interest, as well as characterization of each orf interaction profile. This profile reports the precise engagement onset and termination sequences from which one can infer the decoded codons and the respective residues of the emerging polypeptide chain, as well as on the ribosome speed variations during the interaction7,8. Figure 1 depicts the protocol as a schematic.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62878/62878fig1v2.jpg" /> 
Figure 1: An overview of the SeRP protocol. This protocol can be performed in its entirety within 7-10 days. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3&#39;-Phosphorylated 28 nt RNA control oligonucleotide</td>
			<td>IDT</td>
			<td>custom order</td>
			<td>RNase free HPLC purification; 5&#39;-AUGUAGUCGGAGUCGAGGCGC 
			GACGCGA/3Phos/-3&#39;</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>VWR</td>
			<td>20821</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid phenol&#8211;chloroform</td>
			<td>Ambion</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody: mouse monclonal anti-HA</td>
			<td>Merck</td>
			<td>11583816001</td>
			<td>12CA5</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Roth</td>
			<td>A162.3</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP*</td>
			<td>NEB</td>
			<td>P0756S</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211820</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211699</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto yeast extract</td>
			<td>BD</td>
			<td>212720</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin hydrochloride</td>
			<td>Roth</td>
			<td>2937.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>102445</td>
			<td></td>
		</tr>
		<tr>
			<td>CircLigase II ssDNA Ligase*</td>
			<td>Epicentre</td>
			<td>CL9025K</td>
			<td>100 U/&#956;L</td>
		</tr>
		<tr>
			<td>Colloidal Coomassie staining solution</td>
			<td>Roth</td>
			<td>4829</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche Diagnostics</td>
			<td>29384100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Biological Industries</td>
			<td>A0879</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC treated and sterile filtered water*</td>
			<td>Sigma</td>
			<td>95284</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose anhydrous</td>
			<td>Merck</td>
			<td>G5767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonate</td>
			<td>Roth</td>
			<td>K028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide*</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA ladder, 10 bp O&#39;RangeRuler*</td>
			<td>Thermo Fisher Scientific</td>
			<td>SM1313</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA loading dye*</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0631</td>
			<td>6&#215;</td>
		</tr>
		<tr>
			<td>DNase I, recombinant</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>RNAse free</td>
		</tr>
		<tr>
			<td>dNTP solution set*</td>
			<td>NEB</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA*</td>
			<td>Roth</td>
			<td>8043</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388.260.</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine solution</td>
			<td>Sigma-Aldrich</td>
			<td>67419-1ML-F</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9516</td>
			<td>15 mg/mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>HN78.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HF Phusion polymerase*</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>HK from S. cerevisiae</td>
			<td>Sigma-Aldrich</td>
			<td>H6380-1.5KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>AppliChem</td>
			<td>A1305</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>33539</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Roth</td>
			<td>CN08</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Roth</td>
			<td>T832.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roth</td>
			<td>CN33.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Linker L(rt)</td>
			<td>IDT</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Roth</td>
			<td>KK36.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Roth</td>
			<td>P030.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>Roth</td>
			<td>T879.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl*</td>
			<td>Invitrogen</td>
			<td>AM97606</td>
			<td>5 M</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;H2O</td>
			<td>Roth</td>
			<td>K300.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-activated Sepharose 4 fast-flow beads</td>
			<td>GE Life Sciences</td>
			<td>17090601</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P 40 substitute</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Roth</td>
			<td>2936.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethyl sulfonyl fluoride</td>
			<td>Roth</td>
			<td>6367</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast gels</td>
			<td>Bio-Rad</td>
			<td>5671034</td>
			<td>10% and 12%</td>
		</tr>
		<tr>
			<td>RNase I</td>
			<td>Ambion</td>
			<td>AM2294</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS, 20%</td>
			<td>Ambion</td>
			<td>AM9820</td>
			<td>RNase free</td>
		</tr>
		<tr>
			<td>Sodium acetate*</td>
			<td>Ambion</td>
			<td>AM9740</td>
			<td>3 M, pH 5.5</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Merck</td>
			<td>S8032-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>9265</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide*</td>
			<td>Sigma</td>
			<td>S2770</td>
			<td>1 N</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>16104</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase-In RNase Inhibitor</td>
			<td>Ambion</td>
			<td>AM2694</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transciptase*</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold*</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase*</td>
			<td>NEB</td>
			<td>M0201L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 2*</td>
			<td>NEB</td>
			<td>M0242L</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE polyacrylamide gel*</td>
			<td>Novex</td>
			<td>EC6215BOX</td>
			<td>8%</td>
		</tr>
		<tr>
			<td>TBE&#8211;urea polyacrylamide gel*</td>
			<td>Novex</td>
			<td>EC68752BOX</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>TBE&#8211;urea polyacrylamide gel*</td>
			<td>Novex</td>
			<td>EC6885BOX</td>
			<td>15%</td>
		</tr>
		<tr>
			<td>TBE&#8211;urea sample buffer*</td>
			<td>Novex</td>
			<td>LC6876</td>
			<td>2&#215;</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Roth</td>
			<td>4855</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris*</td>
			<td>Ambion</td>
			<td>AM9851</td>
			<td>1 M, pH 7.0</td>
		</tr>
		<tr>
			<td>Tris*</td>
			<td>Ambion</td>
			<td>AM9856</td>
			<td>1 M, pH 8.0</td>
		</tr>
		<tr>
			<td>UltraPure 10&#215; TBE buffer*</td>
			<td>Invitrogen</td>
			<td>15581-044</td>
			<td></td>
		</tr>
		<tr>
			<td>* - for library preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gasket and spring clamp , 90 mm,</td>
			<td>Millipore</td>
			<td>&#160;XX1009020</td>
			<td></td>
		</tr>
		<tr>
			<td>ground joint flask 1 L ,</td>
			<td>Millipore</td>
			<td>XX1504705</td>
			<td></td>
		</tr>
	</tbody>
</table>

global identification of co-translational interaction networks by selective ribosome profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

As illustrated in the flow chart of this protocol (Figure 1), cells were grown to log phase, and then collected swiftly by filtration and lysed by cryogenic grinding. The lysate was then divided into two: one for total ribosome-protected mRNA footprints and the other for selected ribosome-protected mRNA footprints, on which we performed affinity purification to pull-down the tagged protein-ribosome-nascent chains complexes. We ensured tagged protein expression and the success of the pull-dow...

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Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

Co-translational interactions play a crucial role in nascent chain modifications targeting folding and assembly pathways. Here, we describe selective ribosome profiling, a method for in vivo direct analysis of these interactions in the model eukaryote Saccaromyces cerevisiae. Selective ribosome profiling is the only method to date that captures and characterizes co-translational interactions in vivo in a direct manner.Selective ribosome profiling enables global profiling of any factors and interactions with translating ribosomes in near-codon resolution. Begin by collecting the constructed yeast cells containing the desired tagged proteins. Perform cell lysis using cryogenic grinding in a mixer mill twice for two minutes at 30 Hertz.Centrifuge for two minutes at 30, 000 times G at four degrees Celsius to clear the lysate, and collect the supernatant. Transfer 200 microliters of the supernatant into a microcentrifuge tube for total RNA analysis and 700 microliters into a microcentrifuge tube for immuno-purification. Digest the previously separated total RNA sample using 10 units of RNase I for 25 minutes at four degrees Celsius on a rotating mixing rack at 30 rotations per minute.Prepare the sucrose cushion master mix as described in the text manuscript. Then, load the sample onto 400 microliters of the sucrose cushion and centrifuge for 90 minutes at 245, 000 times G and at four degrees Celsius. Remove the supernatant quickly with a vacuum pump and overlay pellets with 150 microliters of lysis buffer.Secure the sample with paraffin wrap and resuspend the pellets by shaking for one hour at four degrees Celsius at 300 rotations per minute. Wash 100 to 400 microliters of the affinity binding matrix per sample three times with one milliliter of lysis buffer by resuspending the affinity matrix in the lysis buffer. Then, rotate at 30 rotations per minute with a rotating mixing rack at four degrees Celsius for five minutes.Precipitate by centrifugation. Discard the upper liquid and repeat this process three times. Add affinity binding matrix to the previously separated immuno-purification sample and digest it using RNase I.Rotate for 25 minutes at 30 rotations per minute and four degrees Celsius with a rotating mixing rack to bind the protein to the affinity matrix.Prepare the wash buffer master mix as described in the text manuscript. Wash the affinity binding matrix with one milliliter of wash buffer for approximately one minute, rotating in the mix rack. Next, precipitate by centrifugation at 3000 times G for 30 seconds at four degrees Celsius and discard the upper liquid.Repeat three times. Wash twice more in one milliliter of wash buffer, each time for five minutes, rotating in the mix rack. Precipitate again by centrifugation.Use 50 microliters of beads for protein elution with the same amount of 2X sample buffer. Centrifuge to pellet the beads and discard the upper liquid. Elute with 700 microliters of 10-millimolar Tris.Then, freeze in liquid nitrogen. Remove the sample from liquid nitrogen and store at minus 80 degrees Celsius to be used for subsequent RNA extraction. Assess the success of the affinity purification step by Western blot or Coomassie staining with aliquots of each step using mock IP on a non-tagged, wild-type strain as a control for non-specific binding to the affinity matrix.Western blot results after affinity purification demonstrated the success of the tagged protein expression. Isolation of ribosome-protected footprints was validated using the results from the bioanalyzer. While generating a cDNA library for deep sequencing and big data analysis, under-cycling leads to low yield.However, with the dNTPs still present, the reaction proceeds, generating longer PCR artifacts with chimeric sequences due to PCR products priming themselves. The generated library was further validated by high-sensitivity DNA electrophoresis for exact size distribution and quantification. The sequence library was trimmed for adapters and barcodes and the resulting reads were divided into different groups of coding sequences, introns, and intergenic sequences.Ribosome profiles analyzing co-translational interactions of Vma2p with ribosome-associated chaperone Ssb1p, Pfk2p, and Fas1p are shown here with the experimental scheme of each affinity purification. There was ribosome-occupancy along the open reading frame of total translatomes compared to Ssb1, Pfk2p, and Fas1p interactomes. Mean enrichment of Ssb1p, Pfk2p, and Fas1p at each ribosome position along the open reading frame is shown here.This method enables identification and characterization of the various co-translational interactions, ensuring a functional proteome, as well as the study of various diseases. To date, selective ribosome profiling is the only method that can capture and characterize these interactions in a direct manner in vivo.

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Biology

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...