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.

<ol><li>Oh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+ribosome+profiling+reveals+the+cotranslational+chaperone+action+of+trigger+factor+in+vivo%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.</a> Cell. 147 (6), 1295-1308 (2011).</li>
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<li>Matsuo, Y., Inada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ribosome+collision+sensor+Hel2+functions+as+preventive+quality+control+in+the+secretory+pathway%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">The ribosome collision sensor Hel2 functions as preventive quality control in the secretory pathway.</a> Cell Reports. 34 (12), (2021).</li>
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The authors declare no conflicts of interest.

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 manner, in vivo14,15,16.
The first and most critical step is cell collection and lysis. It is imperative to capture, within seconds, ongoing translation, by flash-freezing followed by lysis in a frozen state. Cells collection must be done with haste in order to avoid ribosomal runoff as well as inducing stress translational responses, which can occur rapidly. The second critical step is the affinity purification step. It is imperative to reduce background binding by stringent washing while making sure the co-translational interactions are maintained, which can be facilitated by in vivo cross-linking. As this protocol is based on the highly sensitive NGS (Next Generation Sequencing) high background in the first steps can be amplified in the following cDNA library preparation steps, leading to low signal-to-noise ratios.
Nuclease treatment, to digest all non-protected mRNAs should be evaluated by polysome profiling17 together with careful evaluation of the isolated ribosomal footprints size distribution (as detailed above) to avoid over or under RNA digestion. Calibration of Nuclease concentration and digestion times can facilitate accurate footprint recovery, as over-digestion can lead to ribosomal rRNA digestion, leading to loss of ribosome-protected footprints. It is important to note that under-digestion can also lead to lower discovery rates of ribosome-protected footprints, as the cDNA library preparation steps, as well as data analysis steps described here discard long, uncharacteristic reads.
While rRNA depletion not always constitutes a critical step and is not mandatory, it has some advantages such as cleaner samples and, therefore, a higher rate of genome-mapped reads. On the other hand, there is the possibility of biases, as many rRNA depletion protocols might also cause depletion of the desired ribosome-protected fragments. One should also take into consideration the costs of the rRNA depletion kits. rRNA depletion can be performed after the ribosome-foot print isolation step or after the cDNA circularization step.
cDNA library preparation steps as described here, have been optimized for low mRNA input, as the affinity and ribosome purification steps highly reduce the mRNA input amount, as compared to RNA-seq expression studies. Upscaling the initial amount of cell cultures can greatly facilitate cDNA library generation. Alternatively, any cDNA library protocol of choice can fit with the affinity purification and footprint isolation steps described here. It is important to note that the Nuclease treatment generating the ribosomal footprints requires the resulting mRNA ends repair (cDNA library preparation, Dephosphorylation step) to allow following linker ligation steps in the cDNA protocol described here on in your protocol of choice.
During sequencing, it is important to differentiate SeRP from RNA-Seq, as the generated libraries&#39; heterogeneity greatly varies, depending on the affinity tagged factors. Molecular chaperones and targeting factors are often more promiscuous in binding, interacting with hundreds or thousands of substrates during translation, leading to highly diverse cDNA libraries. However, highly specific interactors, such as co-translational complex assembly interactors can often lead to the generation of much less diverse cDNA libraries. Spike in of diverse and non-diverse libraries on the same lane can greatly improve sequencing and following data analysis results.
Another unique feature of SeRP is its ability to capture local variations in ribosome occupancies along the orf allowing for the discovery of ribosomal shifts in translation rate associated with each set of interactions. It is therefore imperative to compare ribosome occupancies in each codon along the orf to correctly identify enrichment. Utilizing orfs averages can lead to loss of transient interactions or false discovery.
Correct use of the SeRP method opens many co-translational pathways to direct analysis, discovering novel mechanistic features as well as novel ribosome-associated factors, revolutionizing the protein-biosynthesis field.

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><tbody><tr><td>3&#039;-Phosphorylated 28 nt RNA control oligonucleotide</td><td>IDT</td><td>custom order</td><td>RNase free HPLC purification; 5&#039;-AUGUAGUCGGAGUCGAGGCGC&lt;br/&gt; GACGCGA/3Phos/-3&#039;</td></tr><tr><td>Absolute ethanol</td><td>VWR</td><td>20821</td><td></td></tr><tr><td>Acid phenol&amp;ndash;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/&amp;mu;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&#039;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&amp;times;</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 &amp;beta;-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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>KK36.3</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>P030.2</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</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>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</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&amp;ndash;urea polyacrylamide gel*</td><td>Novex</td><td>EC68752BOX</td><td>10%</td></tr><tr><td>TBE&amp;ndash;urea polyacrylamide gel*</td><td>Novex</td><td>EC6885BOX</td><td>15%</td></tr><tr><td>TBE&amp;ndash;urea sample buffer*</td><td>Novex</td><td>LC6876</td><td>2&amp;times;</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&amp;times; 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>&amp;nbsp;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-down by western blot analysis, as can be seen in Figure 2. We validated the isolation of ribosome-protected footprints, which are typically 20-45 nt long by small RNA electrophoresis (2100 BioAnalyzer system), allowing for 5-10 nt shift in size detection, according to the system manual (Figure 3). Then, we generated a cDNA library for deep sequencing and big-data analysis. While generating the cDNA library, note that under-cycling can lead to low yield (as can be seen in lane 2 in Figure 3), but re-amplification is possible in order to recover the generated library. Over-cycling may occur when PCR primers are depleted but the reaction continues. When dNTPs are still present, the reaction proceeds, generating longer PCR artifacts with chimeric sequences due to PCR products priming themselves13 (as can be seen in lanes 3-4 in Figure 3, indicated by the visible smear). If the dNTPs&#39; concentration also becomes limiting, products indicating the presence of heteroduplexes composed of only partially homologous library fragments can appear. Figure 4 acts as a reference, with lane 2 representing optimal amplification, and lane 3 an acceptable amplification. Samples from lanes 4 and 5 (cycles 10 and 11) should not be used due to the possibility of introducing PCR duplicates and artifacts. The generated library was further validated by high sensitivity DNA electrophoresis (the same BioAnalyzer system was used) for exact size distribution and quantification (Figure 5). After 3&#39; end linker ligation, reverse transcription and PCR amplification, a cDNA length distribution as such is expected, with a sharp peak around 175 nt.
We trimmed and removed adapters and barcodes from the sequenced library, and only the reads between 20 and 45 nt were selected for further analysis. Figure 6 shows the resulting length distribution. The reads were divided into different groups of: coding sequences, introns, and intergenic sequences (Figure 7), and further classified as shown in Figure 8.
Final analysis for detection and characterization of co-translational interactions was performed based on the enrichment of ribosome-protected mRNA fragments, producing the graphs in Figure 9. We compared the normalized ribosome occupancy (at each nucleotide along each orf) of the total translatome to its corresponding selected translatome (nascent-interactome). Per nucleotide comparison eliminates translation rates artifacts. Reproducibility between biological replicates was evaluated by Pearson correlation (threshold &#62; 0.6). We present Selective Ribosome Profiles, analyzing co-translational interactions of Vma2p with three proteins: the ribosome-associated chaperone Ssb1p, Pfk2p (Phosphofructokinase) and Fas1p (Fatty acid synthase), with each protein C` terminally tagged by GFP. We performed the protocol in biological replicates. Figure 9 A, D and G shows the experimental scheme of each affinity purification. We next show the ribosome occupancy of total translatomes compared to Ssb1 interactomes along the Vma2p orf, encoding for a subunit of the vacuolar H+-ATPase (Figure 9 B, E and H). Finally, we performed ratio-based ribosome-enrichment profiling (IP/Total) at each ribosome position in [codon/aa] along the orf (Figure 9 C, F and I). Comparing the co-translational interactions of these three proteins with Vma2p, which is being synthesized by the ribosome, revealed that Ssb1 chaperone engages the nascent Vma2p at four different regions along the orf, as we identified four significant enrichment peaks by SeRP. Differently, Pfk2p shows only one significant enrichment peak, as identified by SeRP, in a different position compared to the co-translational chaperone Ssb1. Analysis of Fas1 co-translational interactions with nascent Vma2p did not detect any significant enrichment. Thus, the comparison of these ratio-based enrichment ribosome profiles demonstrates this protocol`s power in detection and characterization of various co-translational interactions in near codon-resolution.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62878/62878fig02.jpg" /> 
Figure 2:&#160;Representative western blot result after affinity purification of BY4741 strain with HA-tagged Naa10. Representative western blot result after affinity purification of BY4741 strain with HA-tagged Naa10 showing a band around 27.8 kDa, while the wild-type, as a negative control, shows no band. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62878/62878fig03.jpg" /> 
Figure 3: Representative BioAnalyzer result after footprint isolation and RNA extraction with acid-phenol:chloroform, and an average size of 25 nt. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62878/62878fig04v2.jpg" /> 
Figure 4: Representative gel-electrophoresis of PCR amplification. Representative gel-electrophoresis of PCR amplification with lanes 2-5 loaded with PCR products from cycles 8-11, and ladders on both sides. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62878/62878fig05.jpg" /> 
Figure 5:&#160;Representative BioAnalyzer result obtained following the creation of a cDNA library. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62878/62878fig06.jpg" /> 
Figure 6:&#160;Expected length distribution of reads after the removal of the adaptors with Cutadapt (removing reads shorter than 20 or longer than 45). <a href="https://www.jove.com/files/ftp_upload/62878/62878fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62878/62878fig07.jpg" /> 
Figure 7: Expected alignment success percentage after removing non-coding RNA reads with Bowtie2 and using TopHat to align the remaining reads to different organisms. The sample was taken from S. cerevisiae (a mutated variant of BY4741). <a href="https://www.jove.com/files/ftp_upload/62878/62878fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62878/62878fig08.jpg" /> 
Figure 8:&#160;A graph generated with RiboToolkit representing expected coding versus non-coding ratio of aligned reads after using Bowtie2 to remove rRNA elements in the reads. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62878/62878fig09.jpg" /> 
Figure 9:&#160;Co-translational interactions of&#160;three different proteins: Ssb1p, Pfk2p, and Fas1p with Vma2p, which is being synthesized by the ribosome, analyzed by SeRP. All y axes are shown in reads per million (RPM) reads.(A, D, G) Experimental scheme of SeRP of Ssb1p, Pfk2p, and Fas1p C` terminally tagged by GFP, respectively. (B, E, H) Ribosome occupancy along the orf of total translatomes compared to Ssb1, Pfk2p, and Fas1p interactomes, respectively (in biological replicates). (C, F, I) Mean enrichment of Ssb1p, Pfk2p, and Fas1p (IP/Total ratio) at each ribosome position in [codon/aa] along the orf, respectively. Variation between biological replicates is indicated by the shaded area. <a href="https://www.jove.com/files/ftp_upload/62878/62878fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 9: 3&#39; Linker and primer sequences. 3&#39; Linker L1: Linker 3-L1 with 5&#697; adenylation and 3&#697; dideoxy-cytidine unique molecular identifiers (&#39;NN...&#39;) (RNase-free HPLC purification; Reverse transcription linker: reverse transcription (L(rt)) with 5&#697; phosphorylated, unique molecular identifiers (RNase-free HPLC purification); PCR forward primer: PCRf; HPLC purified. <a href="https://www.jove.com/files/ftp_upload/62878/Table 9.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File. <a href="https://www.jove.com/files/ftp_upload/62878/Data analysis Supplementary file.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling at JoVE.com

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 ...

global-identification-co-translational-interaction-networks-selective

Nanyang Technological University

Research

JoVE Journal

Biology

2.4K Views.  Technion - Israel Institute of Technology. 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.

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

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Neuroscience

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

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

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

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

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

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

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

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 ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Identification of open reading frames (ORFs), especially those encoding small peptides and being actively translated under specific physiological contexts, is critical for&#160;comprehensive annotations of context-dependent translatomes. Ribosome profiling, a technique for detecting the binding locations and densities of translating ribosomes on RNA, offers an avenue to rapidly discover where translation is occurring at the genome-wide scale. However, it is not a trivial task in bioinformatics to efficiently and comprehensively identify the translating ORFs for ribosome profiling. Described here is an easy-to-use package, named RiboCode, designed to search for actively translating ORFs of any size from distorted and ambiguous signals in ribosome profiling data. Taking our previously published dataset as an example, this article provides step-by-step instructions for the entire RiboCode pipeline, from preprocessing of the raw data to interpretation of the final output result files. Furthermore, for evaluating the translation rates of the annotated ORFs, procedures for visualization and quantification of ribosome densities on each ORF are also described in detail. In summary, the present article is a useful and timely instruction for the research fields related to translation, small ORFs, and peptides.

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level.

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

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mRNA Interactome Capture from Plant Protoplasts

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

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

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data