The authors thank Dr. Percy Tumbale and Dr. Melissa Wells for their critical reading of this manuscript. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS; ZIA ES103247 to R.E.S).

1. Preparation of 7% - 47% sucrose gradients
NOTE: The linear range of the sucrose gradient can be modified to achieve better separation depending on the cell type used. This protocol is optimized for polysome profiles for S. cerevisiae.
<ol>
	<li>Prepare stock solutions of 7% and 47% sucrose in sucrose gradient buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl2, and 1 mM DTT). Filter sterilize the sucrose stock solutions through a 0.22 &#956;m filter and store at 4 &#176;C.</li>
	<li>Prepare 14 mL of 17%, 27%, and 37% sucrose solutions by dispensing and mixing the 7% and 47% sucrose stock solutions in the manner described in Table 1.</li>
	<li>Place six polypropylene centrifuge tubes (14 x 89 mm) into a full view test tube rack. Ensure enough space between the tubes so that actions with one tube do not disturb the others.</li>
	<li>Attach a long needle to a 3 mL syringe. For this protocol, use a 9 in, 22 G needle with a blunt tip (Figure 1), but any needle long enough to reach the bottom of the centrifuge tube will suffice. 
	NOTE: Perform a test fill and dispensation to ensure that the syringe can hold the sucrose solution without any dripping prior to setting up the gradients.</li>
	<li>Add 2 mL of the 7% sucrose to the bottom of each centrifuge tube.</li>
	<li>Add 2 mL of the 17% sucrose beneath the 7% solution by positioning the needle tip within the immediate vicinity of the tube bottom and dispensing the solution slowly and carefully.</li>
	<li>Repeat with 2 mL each of the 27%, 37%, and 47% sucrose solutions. Ensure that each layer is distinguishable from one another by a line marking the separation of densities (Figure 2). 
	NOTE: If the gradients are not going to be used within 48 hours then at this point, prior to their settlement into a continuous gradient, flash freeze the tubes with layered sucrose solutions in liquid nitrogen and store in a -80 &#176;C freezer for long-term storage.</li>
	<li>Store gradients at 4 &#176;C overnight to allow gradients to settle into a continuous, increasing percentage of sucrose. It takes 8-12 h for the layered sucrose solutions to settle into a linear sucrose gradient. Linear gradients are stable for up to 48 h. Overnight storage at 4 &#176;C provides sufficient time for thawing and settlement into a linear gradient for using frozen, layered sucrose solutions. Use a densitometer to assess the gradient quality. 
	NOTE: It is critical to store the gradients in a stable place where they will not be disturbed, as any movements or vibrations will disrupt the gradient.</li>
</ol>
2. Preparation of yeast cell extracts
<ol>
	<li>Inoculate the yeast strain of interest into 50 mL of yeast extract peptone dextrose (YPD) media and grow overnight at 30 &#176;C in a shaking incubator to stationary phase. 
	NOTE: The temperature may vary depending upon yeast strain requirements.</li>
	<li>Transfer 10 mL of stationary phase culture into 1 L of fresh YPD media. Incubate the cells with vigorous shaking at 30 &#176;C (or other suitable temperature) until the culture reaches the mid-exponential growth phase (OD600 = 0.4-0.6).</li>
	<li>At the mid-exponential growth phase, add cycloheximide to the culture at a final concentration of 0.1 mg/mL. Incubate on ice for 5 min.</li>
	<li>Harvest the cells by centrifugation at 3,000 x g for 10 min at 4 &#176;C. 
	NOTE: At this point, cells can be frozen and stored at -80 &#176;C.</li>
	<li>Resuspend the cells in chilled 700 &#956;L of polysome extraction buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% Triton X-100, 0.1 mg/mL of cycloheximide, 0.2 mg/mL of heparin). Add 100 units of RNase Inhibitor and transfer it to a 1.5 mL centrifuge tube.</li>
	<li>Add ~400 &#956;L of pre-chilled glass beads with a size range of 425-600 &#181;m to the centrifuge tube. Disrupt the yeast cells by vigorous agitation in a bead-beater for 5 min.</li>
	<li>Clarify the lysate by centrifugation at 8,000 x g for 5 min at 4 &#176;C.</li>
	<li>Determine the concentration of the RNA in the clarified lysate by measuring the absorbance at 260 nm with a spectrophotometer or using a fluorescence-based RNA detection system. 
	NOTE: For very accurate RNA concentration measurements, use fluorescence-based RNA detection kits.</li>
	<li>Ensure that the RNA concentration is 0.5-1 &#181;g/&#956;L. If the RNA concentration is too low, reduce the volume of polysome extraction buffer used to resuspend the cells in future experiments. 
	&#8203;NOTE: Load the lysate onto the gradients immediately after lysis and RNA quantitation. If necessary, flash freeze the lysates in liquid nitrogen and store at -80 &#176;C for a few days.</li>
</ol>
3. Centrifugation of gradients
<ol>
	<li>Carefully load the lysate onto the top of the gradients. Place the pipette tip against the inner wall, at the top of the polypropylene tube. Gently angle the tube and slowly dispense the lysate onto the top of the gradient by dribbling against the wall. Take great care not to disrupt or disturb the gradient when loading the lysate. 
	NOTE: The amount of lysate loaded will vary per cell type. The content of the RNA will also vary. It may be necessary to perform a number of experiments to determine the amount of lysate needed to generate an optimal polysome profile. For yeast, 300 &#956;g&#160;of RNA is a good starting point for optimization.</li>
	<li>Gently place the tubes into the pre-chilled buckets of a swinging bucket rotor. 
	NOTE: Each polypropylene centrifuge tube should have equal volumes of gradient and the amount of the lysate loaded. This can vary from tube to tube. Ensure that the variation does not cause an imbalance error during ultracentrifugation.</li>
	<li>Centrifuge the gradients at 260,110 x g for 150 min at 4 &#176;C.</li>
</ol>
4. Fraction and data collection
<ol>
	<li>Carefully remove the centrifuge tubes from the swinging bucket rotor and place them in a tube holder.</li>
	<li>Label the 96-well plates to store the fractions and pre-chill on ice. 
	NOTE: Use a 96-well plate suitable for a spectrophotometer that has an optical window down to 230 nm for nucleic acid determinations at 260 nm/280 nm.</li>
	<li>Collect 100 &#181;L or 200 &#181;L fractions starting from the top of the gradient by carefully inserting a pipet tip into the top of the gradient. Collect fractions until the entire gradient is aliquoted. The number of fractions will depend on total gradient volume. 
	NOTE: Collect the fractions in a manner that does not disrupt the rest of the gradient. Ensure that all the fractions have equal volume. In addition to manual fractionation, another low-cost method for fractionation is to use a small peristaltic pump.</li>
	<li>Transfer each fraction to the 96-well plate to reach the bottom of the centrifuge tube. Keep the collected fractions on the ice at all times.</li>
	<li>Measure the absorbance of each fraction at 254 nm with a spectrophotometer. Use the 7% and 47% sucrose solutions as blanks. 
	NOTE: When measuring the absorbance of fractions, bear in mind that for most spectrometers and colorimeters, the most effective absorbance range is 0.1-1. If the absorbance measurements are out of range (&#8805;1.0), the fractions have too much material. Dilute the sample, recollect the data, and then account for the dilution factor when plotting the profile.</li>
	<li>Create the polysome profile by plotting the fraction number versus absorbance.</li>
</ol>

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The authors have nothing to disclose.

Here a method to create polysome profiles without the use of expensive automated fractionation systems has been described. The advantage of this method is that it makes polysome profiling accessible to labs that do not have automated fractionation systems. The major disadvantages of this protocol are tedious hand fractionation and reduced sensitivity compared to the dedicated density fractionation system.
This protocol entails careful preparation of sucrose gradients with resolution sufficient to separate ribosomal subunits, monosomes, and polysomes. When preparing sucrose gradients, it is critical not to introduce air bubbles while loading gradient layers. Air bubbles rise to the top from the bottom and can disrupt the linearity of the gradient. Additionally, the outside of the needle should be wiped before each use, and excess sucrose solution should be wicked off from the lumen to ensure the integrity of each gradient layer. Overnight storage of gradients at 4 &#176;C must be done in a cold room. The vibrations caused by a compressor switching on and off in a refrigerator can disrupt the gradient.
Another critical part of this assay is the volume of the gradient and the concentration of the RNA. The 14 mm x 89 mm centrifuge tubes can hold a volume of 12 mL, but this is the maximum volume that can be accommodated by these tubes and this volume is typically thought of as overfilling these tubes. A good maximum working volume that does not overfill these tubes is 11.5 mL. The sucrose gradient itself has a volume of 10 mL; therefore, the volume of polysome extraction buffer used to resuspend cells should not exceed 1.5 mL. The amount of RNA necessary to generate a good profile will vary by cell type. A number of initial runs should be performed to determine what amount of RNA generates a good polysome profile. Once this amount is determined, it should always be used with that specific cell type to maintain reproducibility and to be able to do comparative analysis. Also, to ensure that the same amount of RNA is loaded each time, the method used for RNA quantitation must always be the same. If a spectrophotometer is used to quantitate RNA, then it should be used at all times. If a fluorometer is utilized, then it should be used at all times. Instruments and quantitation techniques vary widely in both accuracy and sensitivity. Utilizing different instruments or techniques to quantitate RNA experiment to experiment will not generate reproducible results.
This method can be adapted to obtain important information about the status of protein translation in a cell. As mentioned above, the condition of the ribosomal subunits themselves within the assembly process can be determined. Performing experiments in the absence of cycloheximide, which inhibits elongation, enables run-off rate analysis, which indicates whether elongation is altered or not26. The individual fractions are a valuable source of material for further experiments and analysis. For example, the fractions can be used in Northern or Western blotting protocols to identify a specific RNA or protein associated with ribosomal subpopulations. Finally, RNA can be extracted from the fractions and used to identify mRNAs bound to active ribosomes by microarray analysis13,27 or by deep sequencing analysis on a DNA library generated from the total mRNA28.

Ribosomes are mega-Dalton ribonucleoprotein complexes that perform the fundamental process of translating mRNA into proteins. Ribosomes are responsible for carrying out the synthesis of all proteins within a cell. Eukaryotic ribosomes comprise two subunits designated as the small ribosomal subunit (40S) and the large ribosomal subunit (60S) according to their sedimentation coefficients. The fully assembled ribosome is designated as the 80S monosome. Polysomes are groups of ribosomes engaged in translating a single mRNA molecule. Polysome fractionation by sucrose density gradient centrifugation is a powerful method used to create ribosome profiles, identify specific mRNAs associated with translating ribosomes and analyze polysome associated factors1,2,3,4,5,6,7,8,9,10,11,12,13. This technique is often used to separate polysomes from single ribosomes, ribosomal subunits, and messenger ribonucleoprotein particles. The profiles obtained from fractionation can provide valuable information regarding the translation activity of polysomes14 and the assembly status of the ribosomes15,16,17.
Ribosome assembly is a very complex process facilitated by a group of proteins known as ribosome assembly factors18,19,20,21. These factors perform a wide range of functions during ribosome biogenesis through interactions with many other proteins, including ATPases, endo- and exo-nucleases, GTPases, RNA helicases, and RNA binding proteins22. Polysome fractionation has been a powerful tool used to investigate the role of these factors in ribosome assembly. For example, this method has been utilized to demonstrate how mutations in the polynucleotide kinase Grc3, a pre-rRNA processing factor, can negatively affect the ribosome assembly process17,23. Polysome profiling has also highlighted and shown how the conserved motifs within the ATPase Rix7 are essential to ribosome production16.
The procedure for polysome fractionation begins with making soluble cell lysates from cells of interest. The lysate contains RNA, ribosomal subunits, and polysomes, as well as other soluble cellular components. A continuous, linear sucrose gradient is made within an ultracentrifuge tube. The soluble fraction of cell lysate is gently loaded onto the top of the sucrose gradient tube. The loaded gradient tube is then subjected to centrifugation, which separates the cellular components by size within the sucrose gradient by the force of gravity. The larger components travel further into the gradient than the smaller components. The top of the gradient houses the smaller, slower traveling cellular components, whereas the larger, faster traveling cellular components are found at the bottom. After centrifugation, the contents of the tube are collected as fractions. This method effectively separates ribosomal subunits, monosomes, and polysomes. The optical density of each fraction is then determined by measuring the spectral absorbance at a wavelength of 254 nm. Plotting absorbance vs. fraction number yields a polysome profile.
Linear sucrose density gradients can be generated utilizing a gradient maker. Following centrifugation, gradients are often fractionated, and absorbances measured using an automated density fractionation system3,7,13,24,25. While these systems work very well to produce polysome profiles, they are expensive and can be cost-prohibitive to some laboratories. Here a protocol to generate polysome profiles without the use of these instruments is presented. Instead, this protocol utilizes equipment typically available in most molecular biology laboratories.

<table><tbody><tr><td>Automatic Fractionator</td><td>Brandel</td><td></td><td></td></tr><tr><td>Clariostar Multimode Plate Reader</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma Aldrich</td><td>C7698</td><td></td></tr><tr><td>Dithiothreitol</td><td>Invitrogen</td><td>15508-013</td><td></td></tr><tr><td>Glass Beads, acid washed</td><td>Sigma Aldrich</td><td>G8772</td><td>425&amp;ndash;600 &amp;mu;m</td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H4784</td><td></td></tr><tr><td>Magnesium Chloride, 1 M</td><td>KD Medical</td><td>CAC-5290</td><td></td></tr><tr><td>Needle, 22 G, Metal Hub</td><td>Hamilton Company</td><td>7748-08</td><td>custom length 9 inches, point style 3</td></tr><tr><td>Optima XL-100K Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Polypropylene Centrifuge tubes</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Polypropylene Test Tube Peg Rack</td><td>Fisher Scientific</td><td>14-810-54A</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Qubit 4 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33228</td><td></td></tr><tr><td>Qubit RNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32855</td><td></td></tr><tr><td>RNAse Inhibitor</td><td>Applied Biosystems</td><td>N8080119</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S0389</td><td></td></tr><tr><td>SW41 Swinging Bucket Rotor Pkg</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Syringe, 3 mL</td><td>Coviden</td><td>888151394</td><td></td></tr><tr><td>Tris, 1 M,&amp;nbsp; pH 7.4</td><td>KD Medical</td><td>RGF-3340</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td></td></tr><tr><td>UV-Star Microplate, 96 wells</td><td>Greiner Bio-One</td><td>655801</td><td></td></tr></tbody></table>

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.

Three representative polysome profiles are shown in Figure 3. All profiles are from the same yeast strain. A typical polysome profile will have well-resolved peaks for the 40S, 60S, and 80S ribosomal subunits as well as polysomes. The crest of each ribosomal subunit and polysome peak will be apparent on each profile (Figure 3). A representative profile from an automated density fractionation system is shown in Figure 3A. The sucrose gradients used to generate this profile were prepared by hand as described in this protocol. This profile shown in Figure 3A was produced from a continuous absorbance profile as the sucrose gradient was displaced from the bottom up by a chase solution through a detector flow cell and collected in fractions. Because these systems continuously measure absorbance, they can record over 1,500 data points. It is impractical to manually generate the same number of data points. The fraction volume generally utilized for manual profiles is 100-200 &#956;L. A fraction volume within this range yields a profile with enough detail for most comparative analyses. The representative results obtained from both 100 &#956;L and 200 &#956;L fractionations are shown in Figure 3B and Figure 3C. All three profiles presented utilized sucrose gradients prepared as described in this protocol to compare the data yielded. For an example of a polysome profile that utilizes a gradient maker to prepare sucrose gradients as opposed to the manual preparation described in this protocol, a recent manuscript by Chikashige et al. has several examples of gradient profiles generated using an automated gradient maker and fractionation system13.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Final Concentration</td>
			<td>mL of 7% Stock</td>
			<td>mL of 47% Stock</td>
		</tr>
		<tr>
			<td>7%</td>
			<td>14</td>
			<td>0</td>
		</tr>
		<tr>
			<td>17%</td>
			<td>10.5</td>
			<td>3.5</td>
		</tr>
		<tr>
			<td>27%</td>
			<td>7</td>
			<td>7</td>
		</tr>
		<tr>
			<td>37%</td>
			<td>3.5</td>
			<td>10.5</td>
		</tr>
		<tr>
			<td>47%</td>
			<td>0</td>
			<td>14</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of sucrose solutions. Prepare 14 mL of 17%, 27%, and 37% sucrose solutions by dispensing and mixing the 7% and 47% sucrose stock solutions in the volumes indicated.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62680/62680fig01.jpg" /> 
Figure 1: Assembled needle and syringe. A 9 in, 22 G needle with Hamilton needle point style 3 tip attached to a 3 mL syringe via a Luer lock mechanism. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62680/62680fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62680/62680fig02.jpg" /> 
Figure 2: The final appearance of 7%-47% sucrose gradient layers. 7%, 17%, 27%, 37%, and 47% sucrose solutions layered on top of another as described in the protocol. The 17% and 37% layers had blue coloring added to help distinguish the layers for a photograph; no coloring should be added when performing an actual experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62680/62680fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62680/62680fig3v3.jpg" /> 
Figure 3: Polysome profiles. All sucrose gradients used to generate these profiles were prepared using the method described in this protocol. (A) The polysome profile generated by an automated fractionation system (Brandel Fractionator). (B) The polysome profile generated by hand fractionating 200 &#956;L samples described in the current protocol. (C) The polysome profile generated by hand fractionating 100 &#956;L samples described in the current protocol. Polysomes, 40S, 60S, and 80S peaks are indicated for each profile. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62680/62680fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Polysome Profiling without Gradient Makers or Fractionation Systems at JoVE.com

Polysome Profiling without Gradient Makers or Fractionation Systems

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 ...

polysome-profiling-without-gradient-makers-or-fractionation-systems

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.0K Views.  National Institutes of Health. This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Video: Polysome Profiling without Gradient Makers or Fractionation Systems

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 ...

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 ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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

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 ...

Biology

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. ...

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 ...

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 ...

Polysome Profiling without Gradient Makers or Fractionation Systems

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

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Eukaryotic Polyribosome Profile Analysis

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

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