The authors would like to acknowledge John Woolford for the initial basis of this method, TGK is supported by NIH GM057483 and AI076245 and PRC is supported by GM77073.

1. Preparation of 7-47% sucrose gradients
<ol>
	<li>Prepare sucrose gradients one day before use to allow gradients to become continuous. Make sterile 7% and 47% sucrose solutions containing 50mM NH4Cl, 50mM Tris-Acetate pH 7.0 and 12mM MgCl2 and store at 4&deg;C 1.</li>
	<li>For 6 gradients mix the 7% and 47% sucrose stock solutions to make 48 mL of each of the following sucrose concentrations. Add DTT (1M stock) to each sucrose solution to a final concentration of 1mM. 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Final Concentration</td>
 <td>7% sucrose</td>
 <td>47% sucrose</td>
 </tr>
 <tr>
 <td>7%</td>
 <td>48 mL</td>
 <td>0</td>
 </tr>
 <tr>
 <td>17%</td>
 <td>36 mL</td>
 <td>12 mL</td>
 </tr>
 <tr>
 <td>27%</td>
 <td>24 mL</td>
 <td>24 mL</td>
 </tr>
 <tr>
 <td>37%</td>
 <td>12 mL</td>
 <td>36 mL</td>
 </tr>
 <tr>
 <td>47%</td>
 <td>0</td>
 <td>48 mL</td>
 </tr>
</tbody>
</table></li>
	<li>In order to pour the gradients, attach a Pasteur pipette to a 20 mL syringe by securing and sealing with Parafilm or use a long needle. Add 7 mL of 7% sucrose to the bottom of a 1 x 3.5" polyallomer ultracentrifuge tube. Next add 7 mL of 17% sucrose solution under the 7% solution by placing the Pasteur pipette tip near bottom of tube and pipetting no faster than 0.3 mL/sec. Repeat with 7 mL each of 27%, 37% and 47% sucrose solutions. You should observe clear lines between the layers, indicating that minimal mixing has occurred. Store gradients at 4&deg;C overnight along with the rotors, bottles and tubes that will be used to harvest samples.</li>
</ol>
2. Preparation of the yeast extract
<ol>
	<li>Grow 125 mL of yeast culture to an OD600 of 0.8-1.0. Add cycloheximide to the culture to a final concentration of 100 &mu;g/mL and continue shaking at 30&deg;C for 15 min.</li>
	<li>Meanwhile, prepare lysis buffer containing 80 &mu;g/mL cycloheximide, 200 &mu;g/mL heparin, 0.2% DEPC, and 10 mM Tris-HCl pH 7.5, 0.1M NaCl, 30mM MgCl2. Keep everything on ice from this point on.</li>
	<li>Pour the culture into a 250 mL centrifuge bottle and fill with ice. Centrifuge at 8,000 x g (7,250 rpm in a SLA-1500 rotor) for 5 min at 4&deg;C. Wash the pellet once with 10 mL of ice cold lysis buffer and transfer to 15 mL polypropylene tubes. Centrifuge at 5,900 x g (~6,000rpm in a SLA-600TC rotor) for 3 min at 4&deg;C. Resuspend the pellet in 0.5 mL of lysis buffer, transfer to 1.5mL tube and add 0.5 mL of chilled, baked acid-washed glass beads. Vortex the cells for four 30 sec intervals, cooling the tube on ice for 30 sec between each interval. Add another 0.5 mL of lysis buffer into the 1.5 mL tube. Centrifuge at maximum speed in a microcentrifuge (14,000 rpm in an Eppendorf 5417R microcentrifuge) for 10 min at 4&deg;C. Transfer supernatant to a new 1.5 mL microcentrifuge tube. Remove 10 &mu;l to a separate tube to determine the OD260 / OD280 ratio (see step 3.1 below). Store extracts at -80&deg;C.</li>
</ol>
3. Preparation of extracts from mammalian cells
<ol>
	<li>For adherent cells, grow in 100 mm dish to ~70% confluence. You will need one 100 mm dish per 11 ml gradient (typical yield is ~20 OD260 units). Scale the amount linearly for larger or smaller gradient sizes. For non-adherent cells, approximately 1 x 107 cells are needed per 11 ml gradient.</li>
	<li>Prior to lysis, add cycloheximide to 100 &mu;g/mL. Incubate at 37&deg;C for 15 min. Transfer plates to ice and rinse cells twice in ice cold PBS. All future steps MUST be carried out at 0-4&deg;C.</li>
	<li>Remove all traces of PBS by aspiration (let plates drain on an angled bed of ice) and add 1 mL lysis buffer, scrape and transfer to pre-chilled 1.5 mL tube. Lysis buffer components will need to be optimized to the cell type and growth conditions. A good starting buffer would be 20 mM HEPES-KOH, pH 7.4, 15 mM MgCl2, 200 mM KCl, 1% Triton X-100 (v/v), 100 &mu;g/mL cycloheximide, 2 mM DTT, and 1 mg/mL heparin. The key variables are the concentrations of magnesium and potassium chloride as well as the inclusion or exclusion of non-ionic detergents to improve extraction of cytoskeletal or membrane bound polysomes. Lyse cells by passing through 27 gauge syringe needle twice or by 5-10 strokes of dounce homogenization using Type B pestle. The syringe or homogenizer should be pre-chilled prior to use.</li>
	<li>Spin at 14,000 x g for 5 min in refrigerated microcentrifuge. Transfer fresh lysate to the sucrose gradient (for mammalian cells this is made up in lysis buffer lacking Triton X-100 and heparin but otherwise as described in 1.2 and 1.3) or flash freeze in liquid nitrogen for later use.</li>
</ol>
4. Centrifugation of gradients
<ol>
	<li>Thaw polyribosome lysates on ice. Determine the concentration of nucleic acid in the lysate by adding the reserved 10 &mu;L sample to 1 mL of water and reading the OD260/OD280 ratio with a spectrophotometer. Typical yield from a yeast culture grown as described is 50 OD units.</li>
	<li>Using a pipette with the tip placed against the wall of the tube near the surface of the gradient, gently layer 25 OD260 units of the lysate on top of the gradient. For a 35 mL gradient 500 &mu;l of lysate is typically loaded. Lysis buffer can be used to assure all gradients are loaded with an equal volume. Lower amounts of material yield better resolution. Using forceps, carefully lower the gradients into buckets of a swinging bucket rotor.</li>
	<li>Centrifuge the gradients at 100,000 x g (23,000 rpm in a Surespin 630 rotor) for 4 hr at 4&deg;C. This protocol can be adapted for smaller volume gradients.</li>
</ol>
5. Collection of data and fractions
<ol>
	<li>Approximately 30 min prior to the completion of the 4 hr spin attach the needle to the Model 184 tube piercer. Attach the pen to the online Isco Model UA-5 Absorbance/Fluorescence Monitor. Power on the absorbance monitor to allow a stable baseline to be reached prior to analyzing samples. Electronic acquisition of the polysome profile can easily be obtained by attaching a DI-148U data acquisition unit (DATAQ Instruments) to the chart recorder. Profiles can then be collected in real time and saved for later annotation using the accompanying WinDaq software.</li>
	<li>Fill the syringe pump with 50mL of Fluorinert using the reverse flow, rapid setting. Make sure that there are no air bubbles present. Connect the hose from the syringe to the tube piercer and briefly turn on the flow of Fluorinert at 3 mL/min in the forward direction to clear the hose of any air bubbles.</li>
	<li>Place a waste beaker or series of tubes if collecting fractions under the hose at the end of the flow cell to collect sample run off.</li>
	<li>Carefully remove the polyallomer centrifuge tubes containing the sucrose gradient from centrifuge rotor and place them on ice (pre-form the wells in the ice with an empty tube to avoid disturbing gradients).</li>
	<li>To analyze cell extracts, place the gradient tube on the tube piercer. Connect the top of the tube to the outlet and secure. Raise the bottom stage to the centrifuge tube so that the needle pierces the bottom of the tube. Once the needle has protruded through the bottom of the centrifuge tube, begin the flow of Fluorinert in the forward direction at 6mL/min.</li>
	<li>Once the Fluorinert has begun flowing into the tube, monitor the movement of the needle on the online absorbance monitor. Once the needle begins to move, turn on the chart movement to a speed setting of 60. The monitor will record the OD254 starting with the free material followed by 40S ribosomal subunit detection and continuing through to polyribosome complexes. If you are collecting fractions for RNA or protein analysis, typically 0.2 min fractions are taken (1 mL). For RNA analysis, fractions should be collected directly into tubes containing Trizol (Invitrogen), Phenol or SDS/proteinase K, depending on the preferred method of extraction.</li>
	<li>When the end of the polyribosome profile has been reached, turn off the flow of Fluorinert to the centrifuge tube and stop the chart movement of the absorbance/fluorescence monitor.</li>
	<li>Retract the Fluorinert from the centrifuge tube into the syringe by beginning the flow in the reverse direction at a rapid rate making sure not to draw sucrose from the tube into the syringe. Unscrew the centrifuge tube from the tube piercer, lower the needle, and remove the tube.</li>
	<li>Repeat steps 5.5 through 5.8 for each sucrose gradient.</li>
	<li>When all sucrose gradients have been analyzed, retract the Fluorinert from the hose back into the syringe. Remove each part of fractionator (tube piercer and flow cell) including the waste hose located at the top and rinse well with water.</li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figure 1" /> Figure 1. Representative trace of ribosome extract prepared from yeast strain BY4741 (mata his3&Delta;1 leu2&Delta;0 met15&Delta;0 ura3&Delta;0) in the presence of cycloheximide. The ribosomal extract was fractionated utilizing a 7 47% sucrose gradient and analyzed using a pump syringe apparatus attached to a UV detector. Peaks containing polyribosomes and 80S, 60S and 40S ribosomes are indicated.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

No conflicts of interest declared.

The information obtained from the polyribosome profile can provide valuable insight into the translational status of the cell. In addition, the status of the assembly of ribosomal subunits themselves can be determined2. For example the presence of halfmers or 80S and larger peaks with a slight shoulder to the right on the profile indicates a bound 40S subunit awaiting 60S subunit joining. Performing the experiment in the absence of any added cycloheximide or other inhibitor of elongation allows for analysis of the rate of run off, which indicates whether or not elongation is altered3. The fractions themselves are a rich source of reagents for the subsequent determination of the association of a specific mRNA or protein with ribosomal subpopulations by Northern or Western blotting of the fractions. The total pool of mRNAs associated with active ribosomes can be determined via an associated microarray4 or deep sequencing analysis5. The protein associations can also be stabilized by the appropriate addition of a cross linking reagent 6.
Polyribosome analysis from mammalian cells is also worth mentioning as a distinct protocol in terms of the apparent difficulties in establishing the conditions required to obtain stable polysomes. The buffer systems reported in the literature are widely variable 7-10, thus there may be a requirement for cell-type specific optimization of the lysis buffer, or it is equally plausible that the buffer system is not as important as keen attention to working with fresh lysates kept at low temperatures. To our knowledge a systematic evaluation of the parameters affecting mammalian polysome formation has not been reported.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

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 ...

Research

JoVE Journal

Biology

52.5K 次观看.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. 本文介绍了协议的真核细胞的核糖体翻译的提取。一旦提取，核糖体是由蔗糖梯度分馏分离到monosomes和多聚核糖体，允许不同的核糖体的人群进行分析。因此，这种方法是研究翻译调控的黄金标准。

视频: Eukaryotic Polyribosome Profile Analysis

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

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 ...

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

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 ...

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.

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. ...

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

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

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

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

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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 ...

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

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

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

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

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

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