Name: rapid in vivo fixation and isolation of translational complexes from eukaryotic cells
Uploaded: 2021-12-25T14:00:00
Duration: 14 min 29 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells at JoVE.com

This work was supported by Australian Research Council Discovery Project grant (DP180100111 to T.P. and N.E.S), National Health and Medical Research Council Investigator Grant (GNT1175388 to N.E.S.) and Research Fellowship (APP1135928 to T.P.). The authors acknowledge the facilities of Microscopy Australia at the Centre for Advanced Microscopy, Australian National University, a facility that is funded by the University and the Federal Government.

The authors declare no conflicts of interest.

Formaldehyde fixation is a convenient and popular method of achieving rapid in vivo crosslinking of biomolecules10,36,45,46,47,48. Compared to the other potential biomolecule targets, successful capture of translational complexes necessitates an immediate fixation during the snap chilling of the cells or other material. Without the undelayed stabilization, there is a potential for different translation-related processes to continue, shifting the complex distribution away from the unperturbed in vivo state49. Compared to the other methods of translational arrest and ribosomal complex stabilization, the swiftness of formaldehyde action across cell membranes and the indiscriminate nature of the crosslinks promise preservation of the maximal diversity of the translation complex intermediates closer to their natively distributed states50.
The approach presented here has been established and optimized in both yeast and mammalian cells, and methods have now been derived by other groups for use across more diverse biological material, such as in whole vertebrates (e.g., zebrafish embryos)10,38,39,49,51,52. Although these works collectively reassure the versatility and broad applicability of the approach, rapid formaldehyde crosslinking of translational complexes can be considered somewhat difficult to transpose to new types of biological material due to the need of optimizations and adjustments.
A foremost requirement to the success of the method is the re-optimization of the concentration of the formaldehyde and the cell collection and disruption technique. Less permeable, small and round yeast cells require much higher (at least, 10-fold) formaldehyde concentration and physical disruption of the fixed cells. In contrast, large and flattened adherent mammalian cells in culture can be easily over-fixed and require gentle handling upon fixation, while the extraction of the fixed complexes can be performed chemically with membrane disruption using detergents. Under-crosslinking may allow less stable or more short-lived intermediates to dissociate or leak into a later state. Over-crosslinking may negatively affect the ability to isolate and study ribosomal fractions and can create selective biases such as deeper depletion of heavy complexes. In our observation, even minor alterations, such as the type of adherent human cells used, can affect the yield of the recovered crosslinked complexes and may require re-optimization of the crosslinking regimen. We can also anticipate that cells with substantially different permeability properties, such as plant cells, will require additional extensive optimization of the fixation conditions52. Yet, it is difficult to imagine a type of biological material that would be entirely incompatible with the approach.
One consideration pertinent to the mammalian fixation protocol is the density and amount of cell material used as input. It is recommended to have the cells continuously growing without re-seeding or other perturbations for at least 2 days to avoid external influences on cellular translation dynamics. Applicable for most cell types, but for the majority of adherent cells consistently achieved confluence levels of no more than 70% will ensure absence of major contact inhibition effects that can negatively and unpredictably affect translation rates.
Another interesting, and potentially uniquely convenient, feature of formaldehyde fixation stemming from its indiscriminate reactivity is the stabilization effect on translational complexes in systems of mixed taxonomy. Bacterial, and even more so translational complexes of mitochondria, chloroplasts and different intracellular parasites, have been notoriously difficult to target with specific translation inhibitors. In contrast, in the TCP-seq data, footprints mapping to the mitotranscriptome are readily observable in the data38,39,50. An interesting subsequent development could be the use of the approach to investigate translation in entire microcommunities, such as in soil, water or gut samples, where reliable rapid translational arrest and complex stabilization with any other means would be problematic.
It should also be mentioned that for the most complicated material (such as hard and/or bulky tissues), nothing prevents the use of formaldehyde stabilization immediately upon cell disruption and material homogenization. This approach is already frequently employed to remove the cell entry delay when stabilizing translational complexes with specific small molecule inhibitors33,53,54,55. Given that formaldehyde fixation has been traditionally used with excellent results for ex vivo/in vitro sample stabilization in applications such as electron microscopy45,56,57,58, we can expect even less negative effects in this case, particularly those associated with the poor extraction of the translational complexes from the thoroughly fixed cells.
Our findings confirm the usability of rapid formaldehyde fixation to stabilize highly transient complexes, such as those that include eIF4A. It is noteworthy that in contrast to mammals, yeast eIF4A is much more weakly associated with the cap binding complex eIF4F and, as a result, translational complexes in general. eIF4A is usually lost during any extensive purification of the ribosomal material in yeast29,59,60,61,62,63. Yet, in the in vivo-fixed yeast material, it is possible to achieve reliable enrichment of eIF4A in all fractions of translational complexes where its presence would be anticipated. The previously published Sel-TCP-seq data have demonstrated the enrichment of eIF2 and eIF3 that more strongly associate with the ribosomes (but also revealed transiently occurring co-translational protein complex assembly)39. Thus, the method is suitable for the detection of both, stronger and weaker attached constituents of the translational complexes.
To summarize, we have presented an approach useful to gain insights primarily into the changes occurring at the initiation phase of translation and when minimally perturbed ribosomal distribution over the mRNA is required. Importantly, the approach is suitable for the stabilization of relatively labile and dynamic components of translational complexes, such as eIF4A, and can be used broadly subjected to necessary optimizations. We have also provided evidence of the usefulness of formaldehyde fixation in the scenarios of rapid dynamic change of translation, opening up areas of investigation such as fast-paced cellular responses to environmental changes or stress conditions.

Living organisms are subject to dynamic intra- and extracellular changes across their lifespans, which require rapid responses to maintain&#160;homeostasis and ensure survival. To allow environmental adaptation, eukaryotic cells adjust their metabolism via gene expression control. Gene expression control can be exerted during transcription and/or translation; with translational responses generally occurring more rapidly1,2,3,4. For example, translational changes typically arise within 1-30 min of the stress onset, while transcription-level alterations follow hours after stress exposure3,4,5. Alterations to translation output are achieved more rapidly due to the persistent availability of messenger (m)RNA molecules in the cytoplasm. Conversely, at the transcription level, new mRNA molecules must be synthesized, and in eukaryotes, processed and exported from the nucleus, producing extensive delays in the response time2,4,6,7,8.
Acute translational response to stress is generally characterized by an overall decrease in translation output, with the selective upregulation of proteins necessary for cell survival1,3,4,9. Decreasing the protein production output is thought to be crucial due to the high energy expense of the process3,7. To facilitate the selective inhibition and upregulation, translational responses are served by a range of complex regulatory mechanisms. Regulation can be exerted across all phases of translation: initiation, elongation, termination of polypeptide biosynthesis and ribosomal recycling10,11,12,13, but is exhibited most strongly at the initiation phase5,7,9,10,13. During initiation, the small ribosomal subunit (SSU), assisted by eukaryotic initiation factors (eIFs), binds to, and scans the 5&#39; untranslated region (UTR) of mRNA until a start codon is recognized2,5,6,8,11,12,13. Regulatory mechanisms often target eIFs affecting attachment, scanning, and start codon recognition. For example, the initiation factor eIF2, an essential translation factor that aids in the recruitment of an initiator Met-tRNAiMet to the SSU, is often targeted in eukaryotes under stress conditions4,6,11. In yeast, phosphorylation of this factor can be induced under nutrient deprivation and osmotic stress1,4,11,14,15, and in mammalian cells, amino acid starvation, endoplasmic reticulum (ER) stress, UV stress, viral infection, and altered oxygen levels may trigger this response8,9,11. Rapid upregulation of specific mRNA translation is evident in the mammalian cell response to hypoxia, which exhibits a global rapid translation inhibition and selective upregulation of hypoxia-inducible factors (HIFs) biosynthesis. HIFs are transcription factors, which then elicit longer term cellular reprogramming at the DNA transcription level8,9,16. Similar responses have been observed in yeast under heat stress, with rapid translational expression of Heat Shock Proteins (HSPs) followed by delayed transcription-level responses17,18. In addition to nutrient deprivation and heat shock, translational responses in yeast have been studied under varying oxygen8,19, salinity5, phosphate, sulfur20,21 and nitrogen22,23 levels. This research has widespread implications for the industrial uses of yeast, such as baking and fermentation24,25. Translational responses may also be instrumental in furthering understanding of diseases such as neurodegenerative disorders and heart disease, that are characterized by intracellular stresses like oxidative stress. Overall, translational responses are integral to the gene expression control and facilitate rapid adaptation to a broad range of stress conditions in eukaryotic organisms.
In order to study translational responses, methods are required that provide minimally distorted snapshots of the translation landscape. Polysome profiling is a classical approach used in the study of translation across mRNA, involving the separation of poly(ribo)somal fractions of mRNA via ultracentrifugation through sucrose gradients26,27. The approach may be used to explore levels of translation for individual mRNAs (with the detection methods such as reverse transcription and polymerase chain reaction, RT-PCR26), or globally in conjunction with high-throughput techniques (microarray or RNA-seq28,29). A more evolved approach is ribosome profiling, that allows the study of positions of elongating ribosomes along an mRNA molecule at a genome-wide scale, as well as the inference of efficiency of translation across transcriptome and utilization of the main and alternative start sites30,31. Ribosome profiling involves the isolation and sequencing of mRNA fragments protected by ribosomal presence over them. Ribosome profiling has provided considerable insight into translation dynamics across a number of conditions, including hypoxic stress, heat shock and oxidative stress31,32. The technique has been adapted to multiple source material types, including yeast and mammalian cells.
While polysome and ribosome profiling have been fundamental in extending the capabilities of research in translation, the process of translation includes various translational intermediates and complexes that are difficult to capture with these methods11,13. An additional limitation stems from the lack of ability to study rapid response types, as translational complexes are either stabilized in vivo by the addition of specific translation inhibitors (antibiotics), leading to certain ribosome distribution artifacts, or ex vivo upon cell lysis specifically (antibiotics) or unspecifically (high salt or magnesium ions), leading to the deprivation of the shorter-lived or less stable intermediates33,34,35.
Formaldehyde is widely used to crosslink nucleic acids and proteins, such as in chromatin immunoprecipitation (ChIP) and crosslinking immunoprecipitation (CLIP) studies. Its small size and excellent cell permeability allow for a rapid in vivo action36. Based on the rapid formaldehyde crosslinking, the ribosome profiling approach has been extended with the Translation Complex Profile Sequencing (TCP-seq)10,36,37,38,39,40. TCP-seq, first developed in yeast, allows the capture of all translation intermediates, including scanning or post-termination SSU complexes and multiple ribosomal configurations37,38,41,42. The method has been utilized in several studies10,38,39,41,42, some of which use a combinatory approach of both translation inhibitors and formaldehyde crosslinking to facilitate the arrest of translation. A further modified version of the technique, selective TCP-seq39, has recently been employed to include immunopurification of the crosslinked complexes, broadening the scope of the TCP-seq applications. The rapid, efficient and reversible nature of formaldehyde crosslinking makes these approaches suitable for studying transient mRNA:translation complex interactions, particularly in the context of highly dynamic translation-level response pathways.
Here we detail the processes of in vivo formaldehyde crosslinking for the purpose of comprehensive translation complex stabilization and isolation. We provide separate protocols nuanced for yeast and mammalian cells (Figure 1). We further outline examples of the subsequent use of the crosslink-stabilized material (Figure 1), such as for co-purified protein factor detection using immunoblotting (western-blotting), immuno-assisted purification (or &#39;immunoprecipitation&#39;; IP) and enrichment of translational complexes containing specific factors of interest, electron microscopy and RNA sequencing.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62639/62639fig01.jpg" /> 
Figure 1: Schematic depicting an overview of the typical experimental setup. Main steps of in vivo formaldehyde stabilization of translational complexes are depicted as a flowchart, supplemented by information about the key necessary instruments. Potential downstream applications of the crosslinked material are outlined, including examples which have been successfully employed but not directly covered in this protocol, such as SPRI bead purification of RNA, RNA sequencing, and mass-spectrometry. <a href="https://www.jove.com/files/ftp_upload/62639/62639fig01large.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>Yeast extract</td>
			<td>Merck, Sigma-Aldrich</td>
			<td>70161</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck, Sigma-Aldrich</td>
			<td>70178</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose (Dextrose)</td>
			<td>Merck, Sigma-Aldrich</td>
			<td>49139</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine sulphate</td>
			<td>Amresco</td>
			<td>0607-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Merck Sigma-Aldrich</td>
			<td>F11635-500ML</td>
			<td>ACS reagent, 37&#160;wt. % in H2O, contains 10-15% Methanol as stabiliser (to prevent polymerisation)</td>
		</tr>
		<tr>
			<td>RNaseOUT&#8482; Recombinant Ribonuclease Inhibitor</td>
			<td>Invitrogen&#8482; byThermo Fischer Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete&#8482;, EDTA-free Protease Inhibitor Cocktail</td>
			<td>COEDTAF-RO Roche by Merck</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid solution</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion&#8482; RNase I, cloned, 100 U/&#181;L</td>
			<td>Ambion</td>
			<td>AM2294</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase&#8226;In&#8482; RNase Inhibitor (20 U/&#956;L)</td>
			<td>Invitrogen&#8482; by Thermo Fisher Scientific</td>
			<td>AM2694</td>
			<td></td>
		</tr>
		<tr>
			<td>Acidic phenol:chlorophorm:isoamyl alcohol 125:24:1 (pH 4.0-5.0)</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>P1944-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads&#8482; Goat Anti-Mouse IgG</td>
			<td>Invitrogen&#8482; by Thermo Fisher Scientific)</td>
			<td>11033</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M), pH 5.5</td>
			<td>Invitrogen&#8482; by Thermo Fisher Scientific)</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen (5 mg/ml)</td>
			<td>Invitrogen&#8482; by Thermo Fisher Scientific)</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Merck; Sigma Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham&#8482;&#160;Hybond&#174; P Western blotting membranes, PVDF</td>
			<td>Merck</td>
			<td>GE10600023</td>
			<td>PVDF membrane for western blotting</td>
		</tr>
		<tr>
			<td>Bolt&#8482; 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel</td>
			<td>Invitrogen&#8482; by ThermoFischer Sientific</td>
			<td>NW04120BOX</td>
			<td>Protein gel</td>
		</tr>
		<tr>
			<td>4X Bolt&#8482; LDS Sample Buffer</td>
			<td>Invitrogen&#8482; by ThermoFischer Sientific</td>
			<td>B0007</td>
			<td>&#160;LDS sample loading buffer</td>
		</tr>
		<tr>
			<td>Precision Plus Protein&#8482; Kaleidoscope&#8482; Prestained Protein Standards</td>
			<td>BioRad</td>
			<td>1610375</td>
			<td>Protein ladder</td>
		</tr>
		<tr>
			<td>20X Bolt&#8482; MES SDS Running Buffer</td>
			<td>ThermoFischer Scientific</td>
			<td>B0002</td>
			<td>PAGE runninjg buffer</td>
		</tr>
		<tr>
			<td>Intercept&#174; (PBS) Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-70001</td>
			<td>Odyssey Blcoking buffer (PBS)</td>
		</tr>
		<tr>
			<td>IRDye&#174; 800CW Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>92632210</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye&#174; 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>92632211</td>
			<td></td>
		</tr>
		<tr>
			<td>TAP Tag Polyclonal Antibody</td>
			<td>Invitrogen&#8482; by ThermoFischer Sientific</td>
			<td>CAB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-beta Actin antibody</td>
			<td>Abcam</td>
			<td>ab8227</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>84097</td>
			<td>BioUltra, for molecular biology, &#8805;99.5% (HPLC)</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol solution</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>43816</td>
			<td>BioUltra, for molecular biology, ~1 M in H2O</td>
		</tr>
		<tr>
			<td>Terumo Syringe 1CC/mL</td>
			<td>Terumo Syringe</td>
			<td>878499</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>60128</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>(Merck/Sigma-Aldrich)</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>12003C</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline with Calcium and magnesium</td>
			<td>Sigma-Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>10812846001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grinding jar</td>
			<td>Retsch</td>
			<td>02.462.0059</td>
			<td></td>
		</tr>
		<tr>
			<td>MM400 mixer mill</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient Fractionator</td>
			<td>Brandel</td>
			<td>BRN-BR-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer R</td>
			<td>Eppendorf</td>
			<td>Z605271</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5-ml microcentrifuge tubes with locking devices</td>
			<td>Eppendorf Safe-Lock</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Gel Tank</td>
			<td>(Thermo Fisher Scientific)</td>
			<td>A25977</td>
			<td>PAGE running tank</td>
		</tr>
		<tr>
			<td>5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm</td>
			<td>Beckman-Coulter</td>
			<td>344057</td>
			<td></td>
		</tr>
		<tr>
			<td>13.2 mL, Certified Free Open-Top Thinwall Polypropylene, 14 x 89mm - 50Pk</td>
			<td>Beckman-Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ultrafiltration devices</td>
			<td>Merck</td>
			<td>UFC5030</td>
			<td>Ultracel-30 regenerated cellulose membrane, 0.5 mL sample volume</td>
		</tr>
		<tr>
			<td>Thermo Sorvall Evolution RC Floor Super Speed Centrifuge</td>
			<td>Cambridge Scientific</td>
			<td>15566</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter Optima L-90K</td>
			<td>GMI</td>
			<td>8043-30-1191</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc EasYFlask 175cm2</td>
			<td>Thermofisher Scientific</td>
			<td>159910</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Thermofisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological Pipette</td>
			<td>Sigma-Aldrich</td>
			<td>SIAL1250</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>Sigma-Aldrich</td>
			<td>SIAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA lobind tubes</td>
			<td>Eppendorf</td>
			<td>30108051</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold Centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>EP022628188</td>
			<td>for 50 mL tubes</td>
		</tr>
		<tr>
			<td>Orbital Shaking Incubator</td>
			<td>Ratek</td>
			<td>OM11</td>
			<td></td>
		</tr>
		<tr>
			<td>Frezco 17 Microcentrifuge</td>
			<td>Thermofisher Scientific</td>
			<td>75002402</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA lo-bind tubes</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>EP0030108051</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#174; Protein LoBind tubes</td>
			<td>Merck/Sigma-Aldrich</td>
			<td>EP0030108116</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging bucket rotor</td>
			<td>Beckman-Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell&#8482; 150i CO2 Incubator, 150 L</td>
			<td>Thermofisher Scientific</td>
			<td>51026282</td>
			<td></td>
		</tr>
		<tr>
			<td>0,3 mL ultra-fine II short insulin syringe</td>
			<td>BD Medical</td>
			<td>328822</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe with Luer Lok tip</td>
			<td>BD Medical</td>
			<td>302113</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 16 mm Hypodermic Needle</td>
			<td>Terumo</td>
			<td>TUAN2516R1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells at JoVE.com

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 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 ...

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