P. A. is a recipient of a scholarship &#34;Pierre Durand&#34; from the Faculty of medicine of Laval University. This work was supported by the Natural Sciences and Engineering Research Council of Canada (MOP-CG095386) to R. M. The polysome fractionator was acquired through a Canadian Foundation for innovation grant (MOP-GF091050) to R. M. R. M holds a new CIHR investigator salary award.
We are grateful to Drs. E. Khandjian, I. Gallouzi, S. Di-Marco and A. Cammas for helpful advice.

The protocol follows the guidelines approved by Laval&#39;s Ethical Review Board.
1. Preparation of Cell Cultures and Brain Manipulation
<ol>
	<li>Mammalian and Drosophila&#160;Cells
	<ol>
		<li>Grow HeLa cervical cancer cells and Schneider Drosophila embryonic cells as recommended by the American Type Culture Collection. Work with cells at a low passage.</li>
		<li>Plate cells in order to reach 80% confluence the day of the experiment. For best results, use &#8764;12 x 106 of cells for each experimental condition. Before making extracts for polysome analysis, stimulate translation by adding fresh complete medium for at least 90 min.</li>
	</ol>
	</li>
	<li>Brain isolation: Isolate brains from mice aged between 9 and 16 days after birth. Euthanasia involves CO2 inhalation after anesthesia and cervical dislocation. Following sacrifice, isolate the whole brain and either place it at -80 &#176;C or directly transfer it into cold PBS 1X for immediate use.</li>
</ol>
2. Preparation of the Density Gradient Fractionation System
<ol>
	<li>Wash the tubing system with 0.1% SDS for 5 min.</li>
	<li>Wash the tubing system with 70% Ethanol for 5 min, then with RNaseZAP Solution for 5 min&#160;before washing it with DEPC water.</li>
	<li>Pump air in order to dry the tubing system of the apparatus.</li>
</ol>
3. Preparation of the Sucrose Gradients
<ol>
	<li>Make 15% and 55% sucrose solutions in 20 mM Tris-HCl pH 7.4, 1.25 mM MgCl2, 150 mM NaCl, and 1 mM DTT.</li>
	<li>Generate the linear 15-55% sucrose gradient using an Isco Model 160 gradient former, as described by the manufacturer&#39;s instructions. However, it is important to control well the TRIS peristaltic pump speed in order to avoid creation of bubbles or turbulences in the gradients. Keep the gradients at 4 &#176;C until use.</li>
	<li>During the time when the gradient maker program is running, cool the ultracentrifuge to 4 &#176;C.</li>
</ol>
4. Preparation of Cell and Mouse Brain Extracts
<ol>
	<li>Preparation of Cell Extracts
	<ol>
		<li>Place plate(s) on ice and wash cells 3x&#160;with cold PBS 1X.</li>
		<li>Harvest cells (&#8764;12 x 106) in 1 ml lysis buffer (20 mM Tris-HCl at pH 7.4, 1.25 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1% Nonidet P40, 5 U/ml RNase inhibitor, supplemented with complete mini EDTA-free protease inhibitor cocktail tablets), transfer them to an Eppendorf tube, and mix well by passing them 15x&#160;through a 1cc U100 Insulin Syringe 28 G 1/2. Let the cell lysate rest on ice for 15 min.</li>
		<li>Put few drops of cell lysate onto a slide to assess cellular lysis by observing at a phase contrast microscope using a 10X objective. Only nuclei should be visible and no cell membranes should be visible attesting for cell lysis. As a control, observe unlysed cells.</li>
		<li>Clarify the cell lysate by centrifugation at 11,000 x g for 20 min at 4 &#176;C and keep the soluble lysate containing the polyribosomes.</li>
	</ol>
	</li>
	<li>Preparation of Mouse Brain Extracts
	<ol>
		<li>Homogenize the whole isolated brain (&#8764;500 mg) by 10 strokes in an ice-cold Dounce homogenizer with 2 ml of lysis buffer and clarify the homogenate by centrifugation at 11,000 x g for 15 min at 4 &#176;C.</li>
		<li>Load the resulting supernatant onto a cushion of 50% sucrose and proceed with sedimentation for 2 hr at 200,000 x g using an ultracentrifuge rotor TH-641 at 4 &#176;C.</li>
		<li>Resuspend the translucent pellet containing the polyribosomes in 1 ml of lysis buffer and mix well by pipetting up and down. Let the suspension rest on the ice for 30 min&#160;before loading onto gradients.</li>
	</ol>
	</li>
</ol>
5. Loading the Extracts onto Sucrose Gradients and Ultracentrifugation
<ol>
	<li>Measure the concentration of RNA present in the cytoplasmic extract using a spectrophotometer. Carefully and slowly load &#8764;20 OD260 units of the extract onto the 15% to 55% sucrose gradient. Make sure that there is 2-3 mm of available space at the surface of tube to avoid overflowing the tubes during centrifugation.</li>
	<li>Centrifuge the gradients using an ultra-centrifuge for 2 hr 30 min at 230,000 x g using an ultracentrifuge rotor TH-641 at 4 &#176;C. When centrifugation ends, remove the tubes and place them on ice carefully not to disturb the gradients.</li>
</ol>
6. Fractionation of Cytoplasmic Extracts for Polysomes Profiling
<ol>
	<li>Place sucrose gradients on Automated Density Fractionation System and proceed with automated fractionation and collection of fractions (0.5 ml each), as described by the manufacturer.</li>
	<li>Collect each fraction (&#8764;500 &#181;l) into individual Eppendorf tubes with continuous monitoring of absorbance at 254 nm. In parallel of the gradient fractionation operation, the polyribosomal profile will be editing on the chart paper. Alternatively, use a data acquisition unit attached to the chart recorder to have an electronic acquisition of the polysome profile.</li>
	<li>At the end of each run, transfer collected Eppendorfs on dry ice. At this time, either store collected fractions at -80 &#176;C or directly precipitate protein-RNA complexes as follows.</li>
</ol>
7. Protein Extraction and Analysis
<ol>
	<li>To each collected fraction of sucrose gradient, add 2 volumes of 100% cold ethanol and let RNA-protein complexes precipitate at -20 &#176;C overnight.</li>
	<li>Centrifuge each RNA-protein precipitate at 11,000 x g for 20 min at 4 &#176;C then wash with 70% ethanol. Dry and resuspend the precipitate in SDS-PAGE sample buffer before proceeding with Western blot using specific antibodies (see Figure&#160;2).</li>
</ol>
8. RNA Extraction and Analysis
<ol>
	<li>Precipitate RNA-protein complexes as in section 7.1. Resuspend each RNA-protein precipitate in the lysis buffer containing 0.1% SDS. Digest the protein component of the precipitate with 2 mg/ml Proteinase K for 30 min&#160;in a 55 &#176;C water bath.</li>
	<li>Extract RNA components of the precipitate by adding 1 volume of &#34;phenol: chloroform&#34;, 2 volumes of chloroform, and 0.1 volume of 2 M NaOAc pH 4 and centrifuging at 10,000 x g at 4 &#176;C for&#160;20 min. Precipitate RNA from the resulting aqueous phase of each sample overnight at -20 &#176;C by adding 1 volume of isopropanol and 0.2 &#181;g/&#181;l of glycogen. Spin the precipitate for 1 hr at 10,000 x g at 4 &#176;C. Wash the RNA pellet with 70% cold ethanol.</li>
	<li>Resuspend the RNA pellet into a small volume of RNAse free water. Assess the quantity and quality of RNA using the spectrophotometer.</li>
</ol>

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The authors have nothing to disclose.

The polysome profile analysis on sucrose gradients allows measurement of translation initiation by analyzing the density of polysomes isolated from cells or tissues9,11-14. This technique is the best (if not the unique) approach to measure translation initiation in vivo. It is used to monitor the translational status of growing cells during cell cycle15, and to assess the effects of various types of stress including viral infections, hypoxia13,16, radiation17, and chem...

Eukaryotic cells constantly encounter a range of harmful physiological and environmental stress conditions that require a rapid adaptive cell response. Cell stress response involves a precise balance between anti-survival and pro-survival acting factors. Disrupting this balance can have irreversible consequences leading the development of human pathologies such as cancer and neurodegenerative diseases. During the first step of the stress response, cells activate pro-survival pathways that involve the coordinated control of changes in gene expression at the level of mRNA translation.
mRNA translation in eukaryotes is a complex cellular process that involves coordinated interactions between translation initiation factors (eIFs), specific RNA binding proteins (RBDs), and RNA molecules1. mRNA translation is divided into three distinct phases: initiation, elongation, and termination. Although all three phases are subject to regulatory mechanisms, translational control mechanisms target mostly the initiation phase of translation, which thus constitutes the rate-limiting step of protein synthesis2.
Translation initiation is a highly ordered process that begins with the formation of the eIF2a.GTP.Met-tRNAiMet ternary complex and its subsequent binding to the 40S ribosome subunit, leading to the formation of the pre-initiation complex. The next step is the recruitment of the preinitiation complex to mRNA, which involves the activity of translation initiation factors such as eIF4F and eIF3. The 48S preinitiation complex thus formed undergoes specific conformational changes that enable this machinery to start scanning the 5&#39;-untranslated region of the mRNA until it recognizes the initiation codon AUG. Most of the translation initiation factors are then released and 60S subunits are recruited to form an 80S ribosome complex competent for translation, at which point protein synthesis starts (Figure 1). More than one 80S monosome can be translating the same mRNA at a time producing so called polysomes (or polyribosomes). The density of polysomes on a mRNA reflects the initiation, elongation and termination rates and thus is a measure of the translatability of a particular transcript. However, polysome profile is mainly used to assess changes in mRNA translation at the initiation step. Here we have used a proteasome inhibitor as translation initiation inhibitor. Treatment of cancer cells with this drug induces a stress response characterized by activation of the stress kinase named HRI which phosphorylates the translation initiation factor eIF2a3. Phosphorylation of eIF2a is one of the major events leading to the inhibition of translation initiation in mammalian cells4.

<table><tbody><tr><td colspan="4">Cells</td></tr><tr><td>HeLa cervical cancer cells</td><td>American Type Culture Collection (Manassas, VA; ATCC)</td><td>CCL-2</td><td></td></tr><tr><td>Schneider &lt;em&gt;Drosophila&lt;/em&gt; embryonic cells</td><td>American Type Culture Collection (Manassas, VA; ATCC)</td><td>CRL-1963</td><td></td></tr><tr><td colspan="4">Culture medium and Supplements</td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; Medium</td><td>Sigma-Aldrich</td><td>SO146-500ml</td><td>&amp;nbsp;</td></tr><tr><td>DMEM</td><td>Life technologies</td><td>11995-073</td><td>&amp;nbsp;</td></tr><tr><td>FBS</td><td>Fisher Scientist Scientist</td><td>SH30396-03</td><td>&amp;nbsp;</td></tr><tr><td>Penicillin/streptomycin</td><td>Life technologies</td><td>15140122</td><td></td></tr><tr><td colspan="4">Sucrose solutions</td></tr><tr><td>D-sucrose</td><td>Fisher Scientist</td><td>BP220-212</td><td>&amp;nbsp;</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>49767</td><td>&amp;nbsp;</td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientist</td><td>B3925</td><td></td></tr><tr><td colspan="4">Lysis buffer</td></tr><tr><td>Tris HCl</td><td>Fisher Scientist</td><td>BP153-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2670-100G</td><td></td></tr><tr><td>NaCl</td><td>Tekniscience</td><td>3624-05</td><td>&amp;nbsp;</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D 9779</td><td>&amp;nbsp;</td></tr><tr><td>Nonidet P40 (Igepal CA-630 )</td><td>MJS Biolynx</td><td>19628</td><td>&amp;nbsp;</td></tr><tr><td>SDS</td><td>Tekniscience</td><td>4095-02</td><td>&amp;nbsp;</td></tr><tr><td>RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td><td>Life technologies</td><td>10777-019</td><td>&amp;nbsp;</td></tr><tr><td>Antiproteases (complete, mini, EDTA free)</td><td>Roche</td><td>11,836,170,001</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">RNA Extraction</td></tr><tr><td>Proteinase K</td><td>Life technologies</td><td>AM2542</td><td>&amp;nbsp;</td></tr><tr><td>Phenol:chloroform</td><td>Fisher Scientist</td><td>BP1754I-400</td><td>&amp;nbsp;</td></tr><tr><td>Chloroform</td><td>Fisher Scientist</td><td>C298-500</td><td>&amp;nbsp;</td></tr><tr><td>Glycogen</td><td>Life technologies</td><td>10814-010</td><td>&amp;nbsp;</td></tr><tr><td>Isopropanol</td><td>Acros organics</td><td>327270010</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">Antibodies</td></tr><tr><td>anti-FMRP antibody</td><td>Fournier &lt;i&gt;et al.&lt;/i&gt;, Cancer Cell International, 2010</td><td></td><td>&amp;nbsp;</td></tr></tbody></table>

analysis of translation initiation during stress conditions by polysome profiling

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

As aforementioned, polysome profile allows the analysis of changes of translation initiation under stress conditions. Figure 1 is a simplified view of translation initiation which as described earlier is a multistep process involving an ordered assembly of translation initiation complexes. Under normal growth conditions, translation initiation complexes are converted into polyribosomes whose detection by polysome profile attest for an active translation initiation (Figure 2; Untreated). ...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

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

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

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Biology

18.2K Views.  Laval University. Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Video: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

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 ...

Biochemistry

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

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. ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Neuroscience

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 ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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 ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

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

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Polysome Profiling without Gradient Makers or Fractionation Systems

Eukaryotic Polyribosome Profile Analysis

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

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

An Easy Method for Plant Polysome Profiling

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