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>
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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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