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.bdigitaluss.remotexs.co/files/ftp_upload/62639/62639fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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&amp;nbsp;wt. % in H&lt;sub&gt;2&lt;/sub&gt;O, contains 10-15% Methanol as stabiliser (to prevent polymerisation)</td></tr><tr><td>RNaseOUT&amp;trade; Recombinant Ribonuclease Inhibitor</td><td>Invitrogen&amp;trade; byThermo Fischer Scientific</td><td>10777019</td><td></td></tr><tr><td>cOmplete&amp;trade;, 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>&lt;em&gt;Ambion&amp;trade; RNase I, cloned, 100 U/&amp;micro;L&lt;/em&gt;</td><td>Ambion</td><td>AM2294</td><td></td></tr><tr><td>&lt;em&gt;SUPERase&amp;bull;In&amp;trade; RNase Inhibitor (20 U/&amp;mu;L)&lt;/em&gt;</td><td>Invitrogen&amp;trade; 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&amp;trade; Goat Anti-Mouse IgG</td><td>Invitrogen&amp;trade; by Thermo Fisher Scientific)</td><td>11033</td><td></td></tr><tr><td>Sodium Acetate (3 M), pH 5.5</td><td>Invitrogen&amp;trade; by Thermo Fisher Scientific)</td><td>AM9740</td><td></td></tr><tr><td>Glycogen (5 mg/ml)</td><td>Invitrogen&amp;trade; 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&lt;sup&gt;&amp;trade;&lt;/sup&gt;&amp;nbsp;Hybond&lt;sup&gt;&amp;reg;&lt;/sup&gt; P Western blotting membranes, PVDF</td><td>Merck</td><td>GE10600023</td><td>PVDF membrane for western blotting</td></tr><tr><td>Bolt&amp;trade; 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel</td><td>Invitrogen&amp;trade; by ThermoFischer Sientific</td><td>NW04120BOX</td><td>Protein gel</td></tr><tr><td>4X Bolt&amp;trade; LDS Sample Buffer</td><td>Invitrogen&amp;trade; by ThermoFischer Sientific</td><td>B0007</td><td>&amp;nbsp;LDS sample loading buffer</td></tr><tr><td>Precision Plus Protein&amp;trade; Kaleidoscope&amp;trade; Prestained Protein Standards</td><td>BioRad</td><td>1610375</td><td>Protein ladder</td></tr><tr><td>20X Bolt&amp;trade; MES SDS Running Buffer</td><td>ThermoFischer Scientific</td><td>B0002</td><td>PAGE runninjg buffer</td></tr><tr><td>Intercept&amp;reg; (PBS) Blocking Buffer</td><td>LI-COR</td><td>927-70001</td><td>Odyssey Blcoking buffer (PBS)</td></tr><tr><td>IRDye&amp;reg; 800CW Goat anti-Mouse IgG Secondary Antibody</td><td>LI-COR</td><td>92632210</td><td></td></tr><tr><td>IRDye&amp;reg; 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&amp;trade; 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, &amp;ge;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 H&lt;sub&gt;2&lt;/sub&gt;O</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&#039;s Modified Eagle&#039;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&#039;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&amp;reg; 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&amp;trade; 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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Unit 1

Translation: RNA to Protein

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3.8K Views.  The Australian National University. 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.

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

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

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

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

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 ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

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

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Quantitative Immunofluorescence to Measure Global Localized Translation

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Single-Molecule Imaging of Nuclear Transport

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

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos