This research was funded by CSIC I+D 2020 grant No. 282, FVF 2017 grant No. 210, and PEDECIBA (Mar&#237;a Martha Sainz).

1. Plant growth and rhizobia inoculation
<ol>
	<li>To generate the required nodules, sow the soybean seeds of choice in the selected substrate in a growth chamber under controlled conditions. 
	NOTE: In this protocol, seeds were sown in a 0.5 L plastic bottle filled with a mix of sand:vermiculite (1:1). The growth chamber was set with a day/night cycle temperature of 28 &#176;C /20 &#176;C, respectively, and a light/darkness photoperiod of 16 h/8 h, respectively. The photosynthetically active radiation intensity was 620 &#181;mol&#183;m&#8722;2&#183;s&#8722;1.</li>
	<li>Prepare in advance liquid Yeast-Extract Mannitol (YEM)-medium (see the Table of Materials). Autoclave the medium.</li>
	<li>On the same day of seed sowing, inoculate one flask containing 100 mL of YEM with the Bradyrhizobium elkanii U1302 strain. Incubate the flask at 28 &#176;C on an orbital shaker at 100 rpm.
	<ol>
		<li>Let the rhizobia grow for 2 days and inoculate the seedlings with 2 mL of the culture.</li>
	</ol>
	</li>
</ol>
2. Water deficit treatment (optional)
NOTE: This protocol outlines the water deficit treatment of the soybean plants. This part can be changed or omitted entirely depending on the experimental question at hand.
<ol>
	<li>Grow the soybean seedlings for 19 days (V2-3 developmental stage) without water restriction. Keep the substrate at field capacity with B&#38;D-medium17 supplemented with 0.5 mM KNO3.</li>
	<li>On day 20, withdraw watering. 
	NOTE: Here, the water content was measured daily by gravimetry (water gravimetric content) during the growth and water deficit periods.</li>
	<li>Measure stomatal conductance in triplicate daily on the abaxial leaf surface with a porometer, as instructed by the manufacturer (see the Table of Materials), from day 20 until the end of the water deficit period. 
	&#8203;NOTE: The end of the water deficit period was determined individually for each plant when the stomatal conductance value was approximately 50% of the one obtained on day 20 from sowing.</li>
</ol>
3. Nodule harvest
<ol>
	<li>Collect liquid nitrogen in a clean container and label 15 mL tubes (one for each plant). 
	CAUTION: Handle liquid nitrogen wearing cryo gloves, face shields, and safety goggles.</li>
	<li>Retrieve each plant individually. Cut and discard the aerial part. Thoroughly wash the root with water to remove any remaining substrate.</li>
	<li>Detach each nodule from the root and collect them in a prechilled 15 mL tube. Snap-freeze the tubes and store them at &#8722;80 &#176;C.</li>
</ol>
4. Preparation of cytosolic extracts
NOTE: The final aim of this protocol is to obtain high-quality total RNA (TOTAL) and polysome-associated RNA (PAR). Therefore, work under conditions that prevent RNA degradation, always keeping the samples at 4 &#176;C and using RNase-free laboratory equipment and solutions. Unless specified, all the solutions are prepared with sterile ultrapure water.
<ol>
	<li>Preparation of buffer stock solutions
	<ol>
		<li>Prepare the autoclavable stock solutions listed in Table 1, autoclave them for 15 min, and keep at RT.</li>
		<li>Prepare the filter-sterilizable stock solutions listed in Table 1, filter-sterilize them, and keep them at &#8722;20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare polysome extraction buffer (PEB, see Table 1) and keep it on ice. 
	NOTE: The volumes given are for six samples.</li>
	<li>Cool the centrifuge to 4 &#176;C and place 2 mL microcentrifuge tubes on ice.</li>
	<li>Weigh approximately 0.2 g of intact nodules in a weighing dish and transfer them to a precooled 2 mL tube. 
	NOTE: Perform this step quickly to avoid the thawing of the samples. Alternatively, an estimated nodule volume of 0.4-0.5 mL can be used instead of the 0.2 g.</li>
	<li>Add 1.2 mL of PEB, let the sample thaw for 2 min, and homogenize with a tissue grinder until complete disruption and homogenization of the nodules. 
	NOTE: Be sure to grind the nodules once thawed as they will be soft enough to be easily disrupted with tissue grinders (see the Table of Materials) in microcentrifuge tubes. Frozen nodules are difficult to grind. Also, it is recommended to place the tubes in a benchtop cooler (0 &#176;C) for proper homogenization while keeping samples cool.</li>
	<li>Incubate the samples on ice with gentle agitation for 10 min or until all samples have been processed.</li>
	<li>Centrifuge at 16,000 &#215; g for 15 min at 4 &#176;C to pellet the debris. Recover the supernatant and repeat the centrifuge step.</li>
	<li>Carefully recover the clarified cytosolic extract and transfer a 200 &#181;L aliquot to a clean 1.5 mL microcentrifuge tube for TOTAL isolation (TOTAL samples; see section 7).</li>
</ol>
5. Preparation of sucrose cushions
NOTE: This protocol uses a two-layer sucrose cushion (12% and 33.5%) in 13.2 mL ultracentrifuge tubes (see the Table of Materials). All solutions are prepared with sterile ultrapure water.
<ol>
	<li>Prepare the sucrose and salt stock solutions.
	<ol>
		<li>Prepare 100 mL of 2 M sucrose solution (68.5%, Table 1).</li>
		<li>Prepare 10x salt solution (Table 1).</li>
	</ol>
	</li>
	<li>Prepare the two layers of the sucrose cushion as described in Table 2. See Table 1 for the composition of the antibiotic (CHX and CHL) stock solutions. 
	NOTE: The volumes given are for six cushions.</li>
	<li>Pour 4.5 mL of the 33.5% sucrose layer into the centrifuge tubes. Then, add 4.5 mL of the 12% layer carefully and slowly with a P1000 micropipette. Put the tubes on ice until the addition of the clarified cytosolic extract.</li>
</ol>
6. Polysome purification
<ol>
	<li>Load 1 mL of the clarified cytosolic extract (step 4.8) on top of the sucrose cushion by carefully pipetting onto the sidewall of the tube.</li>
	<li>Transfer the ultracentrifuge tubes to precooled buckets and centrifuge at 217,874 &#215; g&#160;(35,000 rpm in the referenced rotor [see the Table of Materials]) for 2 h at 4 &#176;C.</li>
	<li>Discard the remaining cytosolic extract and sucrose cushion, and resuspend the polysomal pellet with 200 &#181;L of precooled RB (previously prepared according to Table 1).</li>
	<li>Incubate for 30 min on ice and then transfer the polysomal resuspension to 1.5 mL precooled tubes. Proceed with the RNA extraction (PAR samples).</li>
</ol>
7. RNA extraction and quality control
NOTE: This step is performed for TOTAL (step 4.8) and PAR samples (step 6.4).
<ol>
	<li>Prepare 75% EtOH with sterile ultrapure water and cool it to &#8722;20 &#176;C. Additionally, cool chloroform and isopropanol to &#8722;20 &#176;C.</li>
	<li>Homogenize the samples with 750 &#181;L of the RNA isolation reagent and incubate for 5 min at RT.</li>
	<li>Add 200 &#181;L of cold chloroform and shake the tubes vigorously for 15 s. Incubate at RT for 10 min.</li>
	<li>Centrifuge at 12,000 &#215; g for 15 min at 4 &#176;C for phase separation. Transfer 500 &#181;L of the upper aqueous phase to a clean tube without disturbing the pink organic phase, and add 375 &#181;L of cold isopropanol and 0.5 &#181;L of RNase-free glycogen (see the Table of Materials). Mix thoroughly by pipetting up and down. 
	NOTE: Glycogen is a carrier used as a co-precipitant to increase nucleic acid recovery from alcohol precipitation.</li>
	<li>Incubate the mix for 10 min at 4 &#176;C and centrifuge at 12,000 &#215; g for 15 min. Discard the supernatant.</li>
	<li>Wash the RNA precipitate with 1 mL of cold 75% EtOH. Mix by brief vortexing. 
	NOTE: At this point, the samples can be stored at &#8722;20 &#176;C overnight.</li>
	<li>Centrifuge at 7,500 &#215; g for 5 min at 4 &#176;C, carefully remove the supernatant with a micropipette, and air-dry the RNA pellet.</li>
	<li>Dissolve the pellet in 50 &#181;L of RNase-free water and incubate at 65 &#176;C for 5 min.</li>
	<li>Assess the RNA concentration and integrity with highly sensitive capillary electrophoresis18 (see the Table of Materials) and/or electrophoresis on a 2% RNase-free agarose gel19 (see the Table of Materials). 
	NOTE: If the RNA samples are not going to be used immediately or are going to be sent to the sequencing facility for RNA-seq library preparation, it is recommended to use EtOH to precipitate them.</li>
</ol>
8. RNA precipitation
<ol>
	<li>Cool the centrifuge and EtOH to 4 &#176;C.</li>
	<li>Prepare 3 M sodium acetate.</li>
	<li>Prepare 70% EtOH with sterile ultrapure water and cool it to &#8722;20 &#176;C.</li>
	<li>Estimate the sample volume. Add 0.1 volumes of 3 M sodium acetate, 3 volumes of cold EtOH, and 0.5 &#181;L of RNase-free glycogen. Mix thoroughly.</li>
	<li>Leave at &#8722;20 &#176;C until needed. 
	&#8203;NOTE: RNA precipitated samples can be stored for up to 1 year at &#8722;80 &#176;C.</li>
</ol>
9. Standard pipeline for gene expression analysis
<ol>
	<li>Perform an average of 40M Illumina paired-end reads, with read length &#62;100 bp, for confident transcriptome and translatome data analysis. 
	NOTE: Standard RNA-seq data analysis includes the following steps 9.2-9.5.</li>
	<li>Carry out read quality inspection using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)&#160;or the multisample MultiQC20.</li>
	<li>Remove adaptor and low-quality sequences using either Trimmomatic21, BBDuk (http://jgi.doe.gov/data-and-tools/bb-tools/), cutadapt22, sickle23, or Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), among others.</li>
	<li>If a reference genome is available for the species, carry out read mapping against the reference, followed by abundance quantification, using Bowtie224, TopHat225 or Salmon26, and featureCounts27, besides other open source tools.</li>
	<li>Perform statistical analysis for differential expressed genes identification using edgeR28, DESeq229, or limma30, among others. 
	NOTE: These open-source software can be used locally, via the command line. Alternatively, they can be operated through a web browser on a public server, such as Galaxy31 or GEOexplorer32, which offer a graphical user interface, so that no command line knowledge is required for their use.</li>
</ol>

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W., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+polyribosomal+complexes+of+Arabidopsis+for+global+analysis+of+gene+expression.">Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.</a> Plant physiology. 138 (2), 624-635 (2005).</li><li>Mustroph, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+translatomes+of+discrete+cell+populations+resolves+altered+cellular+priorities+during+hypoxia+in+Arabidopsis.">Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18843-11848 (2009).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S., 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 (5924), 218-223 (2009).</li><li>Brar, G. A., 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=Ribosome+profiling+reveals+the+what,+when,+where+and+how+of+protein+synthesis.">Ribosome profiling reveals the what, when, where and how of protein synthesis.</a> Nature Reviews. Molecular Cell Biology. 16 (11), 651-664 (2015).</li><li>Eastman, G., Smircich, P., Sotelo-Silveira, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+ribosome+footprints+to+understand+translation+at+a+genome+wide+level.">Following ribosome footprints to understand translation at a genome wide level.</a> Computational and Structural Biotechnology Journal. 16, 167-176 (2018).</li><li>Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RNA-seq+of+pathogen+and+host.">Dual RNA-seq of pathogen and host.</a> Nature Reviews Microbiology. 10 (9), 618-630 (2012).</li></ol>

The authors have no conflicts of interest.

Studying gene expression regulation at the translational level is critical to better comprehend different biological processes since the endpoint of cell gene expression is protein abundance13,14. This can be assessed by analyzing the translatome of the tissue or organism of interest for which the polysomal fraction should be purified and its associated mRNAs analyzed3,4,34,35,36.
A method is presented here to purify soybean symbiotic nodule polysomes through sucrose cushions, followed by TOTAL and PAR extraction. Obtaining both RNA fractions is one relevant point of this protocol since RNA-seq and standard pipelines for gene expression could be implemented afterward. Thus, the transcriptome and the translatome of the nodule can be studied.
An alternative method for polysome purification is the translating ribosome affinity purification (TRAP) method, in which the expression of an epitope-tagged version of ribosomal protein L18 allows for the immunoprecipitation of ribosomes37,38. However, this method requires the generation of transgenic plants, which can be challenging for some species such as the soybean. Moreover, the ribosome profiling or Ribo-seq method15,39,40,41, which involves the deep sequencing of ribosome-protected mRNA fragments after polysome purification, is an alternative for studying the translatome with higher resolution than polysome profiling, since the exact number and position of ribosomes on individual mRNAs can be assessed. Nevertheless, this approach is more laborious, requires higher amounts of sample, and is computationally intensive, since small RNA fragments are recovered.
There are several critical experimental aspects to consider, such as the quality of the sample from which the polysomes will be purified, especially if frozen tissue is used, as is the case in this protocol. Intact nodules should be detached from the root and immediately frozen in liquid nitrogen before storage at &#8722;80 &#176;C. In our experience, polysomes and their associated mRNAs can be successfully purified from nodules within a few months of storage. Furthermore, the whole protocol must be implemented under conditions that prevent RNA degradation to obtain high-quality TOTAL and PAR, which is critical for downstream applications such as cDNA synthesis and high-throughput sequencing.
A major limitation of the method is the ultracentrifugation step, as ultracentrifuges are not widely available in many laboratories. Also, it is relevant to mention that the pellet obtained after the centrifugation step might contain, besides polysomes, other ribonucleoproteins complexes that are not translationally active. Moreover, one consideration if performing the protocol presented here with another soybean tissue is that it will probably require optimizing the amount of sample, the sample:PEB ratio, and the homogenization procedure.
Considering that the sample used in this protocol is an organ formed due to a symbiotic interaction between a plant root cell and a bacteria, performing a Dual RNA-seq analysis42 (with TOTAL and PAR samples) would be interesting since it would allow examination of the transcriptional and translational responses from both organisms. The method described here could be helpful for this matter if the extraction of the bacterial RNAs is optimized.

Leguminous plants, such as soybean (Glycine max), can establish symbiosis with specific soil bacteria called rhizobia. This mutualistic relationship elicits the formation of novel organs, the symbiotic nodules, on the plant roots. The nodules are the plant organs hosting the bacteria and consist of host cells whose cytoplasm is colonized with a specialized form of rhizobia called bacteroids. These bacteroids catalyze the reduction of atmospheric nitrogen (N2) into ammonia, which is transferred to the plant in return for carbohydrates1,2.
Although this nitrogen-fixing symbiosis is one of the most well-studied plant-microbe symbioses, many aspects remain to be better understood, such as how plants subjected to different abiotic stress conditions modulate their interaction with their symbiotic partner and how this affects nodule metabolism. These processes could be better understood by analyzing the nodule translatome (i.e., the subset of messenger RNAs [mRNAs] actively translated). Polyribosomes or polysomes are complexes of multiple ribosomes associated with mRNA, commonly used to study translation3. The polysome profiling method consists of the analysis of the mRNAs associated with polysomes and has been successfully used to study the posttranscriptional mechanisms controlling gene expression that occurs in diverse biological processes4,5.
Historically, genome expression analysis has focused primarily on determining mRNA abundance6,7,8,9. However, there is a lack of correlation between transcript and protein levels due to the different stages of posttranscriptional regulation of gene expression, particularly translation10,11,12. Moreover, no dependence has been observed between the changes at the level of the transcriptome and those that occur at the level of the translatome13. The direct analysis of the set of mRNAs that are being translated allows a more accurate and complete measurement of the cell gene expression (whose endpoint is protein abundance) than the one obtained when only mRNA levels are analyzed14,15,16.
This protocol describes how plant-derived polysomes are purified from intact soybean nodules by differential centrifugation through a two-layer sucrose cushion (Figure 1). However, since bacteroid-derived ribosomes are also present in the nodules, a mix of ribosomes and RNA species are purified, even though the eukaryotic ones represent the main fraction (90%-95%). The subsequent RNA isolation, quantification, and quality control are also described (Figure 1). This protocol, in combination with RNA-seq, should provide experimental results on the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64269/64269fig01.jpg" /> 
Figure 1: Schematic overview of the proposed methodology for eukaryotic polysome purification from symbiotic nodules. The scheme gives an overview of the steps followed in the protocol from (1) plant growth and (2) nodule harvest to (3) preparation of the cytosolic extracts, (3) obtaining TOTAL samples and (4) PAR samples, and (5) RNA extraction and quality control. Abbreviations: PEB = polysome extraction buffer; RB = resuspension buffer; TOTAL = total RNA; PAR = polysome-associated mRNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64269/64269fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;Plant growth and rhizobia inoculation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Daihan Scientific</td><td></td><td>Model SHO-1D</td></tr><tr><td>YEM-medium</td><td>Amresco</td><td>J850 (yeast extract) 0122 (mannitol)</td><td></td></tr><tr><td>&lt;strong&gt;Water deficit treatment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>221295</td><td></td></tr><tr><td>Porometer</td><td>Decagon Device</td><td></td><td>Model SC-1</td></tr><tr><td>Scalpel</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Preparation of cytosolic extracts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Brij L23</td><td>Sigma-Aldrich</td><td>P1254</td><td></td></tr><tr><td>Centrifuge</td><td>Sigma</td><td></td><td>Model 2K15</td></tr><tr><td>Chloranphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma-Aldrich</td><td>C7698</td><td></td></tr><tr><td>DOC</td><td>Sigma-Aldrich</td><td>30970</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D9779</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Igepal CA 360</td><td>Sigma-Aldrich</td><td>I8896</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1.04936</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Plastic tissue grinder</td><td>Fisher Scientific</td><td>12649595</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>PTE</td><td>Sigma-Aldrich</td><td>P2393</td><td></td></tr><tr><td>Tris</td><td>Invitrogen</td><td>15504-020</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Weighing dish&amp;nbsp;</td><td>Deltalab</td><td>1911103</td><td></td></tr><tr><td>&lt;strong&gt;Preparation of sucrose cushions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sucrose</td><td>Invitrogen</td><td>15503022</td><td></td></tr><tr><td>SW 40 Ti rotor</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman-Coulter</td><td></td><td>Optima L-100K</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman-Coulter</td><td>344059</td><td>13.2 mL tubes</td></tr><tr><td>&lt;strong&gt;RNA extraction and quality control&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Thermo scientific</td><td>R0492</td><td></td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td></td><td>Model 2100. Eukaryote total RNA nano assay</td></tr><tr><td>Chloroform</td><td>DI</td><td>41191</td><td></td></tr><tr><td>Ethanol</td><td>Dorwil</td><td>UN1170</td><td></td></tr><tr><td>Isopropanol</td><td>Mallinckrodt</td><td>3032-06</td><td></td></tr><tr><td>Glycogen</td><td>Sigma</td><td>10814-010</td><td></td></tr><tr><td>TRIzol LS</td><td>Ambion</td><td>102960028</td><td></td></tr><tr><td>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Biologix</td><td>10-0152</td><td></td></tr><tr><td>Filter tips 10 &amp;micro;L</td><td>BioPointe Scientific</td><td>321-4050</td><td></td></tr><tr><td>Filter tips 1000 &amp;micro;L</td><td>BioPointe Scientific</td><td>361-1050</td><td></td></tr><tr><td>Filter tips 20 &amp;micro;L</td><td>BioPointe Scientific</td><td>341-4050</td><td></td></tr><tr><td>Filter tips 200 &amp;micro;L</td><td>Tarsons</td><td>528104</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL</td><td>Tarsons</td><td>500010-N</td><td></td></tr><tr><td>Microcentrifuge tubes 2.0 mL</td><td>Tarsons</td><td>500020-N</td><td></td></tr><tr><td>Sequencing company</td><td>Macrogen</td><td></td><td></td></tr><tr><td>Sterile 250 mL flask</td><td>Marienfeld</td><td>4110207</td><td></td></tr></tbody></table>

polysome purification from soybean symbiotic nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

The quantity and quality assessment of the TOTAL and PAR fractions purified with the abovementioned procedure is key to determining its success, since for most downstream applications, such as RNA sequencing, high-quality samples are fundamental for library preparation and sequencing. Moreover, the integrity of the RNA molecules allows the capture of a snapshot of the gene expression profile at the moment of sample collection18. In this context, an RNA integrity number (RIN) is obtained when performing these measurements using a Bioanalyzer. The RIN is used for assigning integrity values in a robust, reliable, and user-independent way, ranging from 10 (intact) to 1 (totally degraded)18.
Figure 2A,B show the standard output of the high-sensitivity capillary electrophoresis system (Bioanalyzer) for several TOTAL and PAR sample fractions. Very good quality is observed for all the samples since sharp ribosomal RNA (rRNA) bands can be visualized without any smeared appearance. However, this is not reflected in the RIN values, as they range between 5.9 and 7.5, which corresponds to non-intact samples. Nevertheless, as previously mentioned, there is no visual sign of sample degradation, as can be seen in Figure 2C. Figure 2D shows an example of a bioanalyzer result of an almost entirely degraded sample for comparison. The equipment also failed to identify the 18S and 25S peaks, as seen in the electropherograms both for TOTAL and PAR samples (Figure 2B). Consequently, the proportion of the ribosomal bands (25S:18S; another indicator of RNA integrity) could not be calculated. High-quality RNA usually exhibits a 25S:18S 2:1 ratio33.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64269/64269fig2v2.jpg" /> 
Figure 2: Total RNA and polysome-associated mRNA quantity and quality assessment. (A)&#160;Representative virtual gel reconstruction of high-sensitivity capillary electrophoresis (Bioanalyzer) results from 12 samples with their respective RIN and concentration values. Lane 1 to lane 6 correspond to total RNA (TOTAL) fractions of six nodule samples, and lane 7 to lane 12 correspond to polysome-associated mRNA (PAR) fractions of the same samples. Green line: lower marker present in all lanes. (B) Electropherogram representation is shown for sample 4 (as an example of TOTAL) and sample 10 (as an example of PAR). 18S and 25S peaks are indicated. Peaks possibly corresponding to 16S and 23S (prokaryotic rRNAs) are indicated with *. Zoom-ins on these peaks are shown (B1 and B2). The Eukaryote Total RNA Nano assay was used in the Bioanalyzer. (C) Representative agarose gel electrophoresis results from six samples (left): 10 &#181;L of TOTAL and PAR fractions were loaded on a 2% agarose gel and separated (80 V for 35 min). On the right, there is another gel of sample 2 only, where the prokaryotic rRNAs can be visualized more clearly. Eukaryotic rRNA (18S and 25S) are indicated by black arrowheads and prokaryotic rRNAs (16S and 23S) by grey arrowheads.(D) Example of a Bioanalyzer result of a degraded sample. L: standard ladder. Green line: lower marker present in all lanes. Abbreviations: RIN = RNA integrity number; PAR = polysome-associated mRNA; nt = nucleotides; L = standard ladder. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64269/64269fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Since these samples are from symbiotic nodules where eukaryotic and prokaryotic RNAs coexist, the purified RNA is a mix of both species (although the eukaryotic ones are the majority). Hence, it is expected to observe peaks corresponding to 16S and 23S rRNA in the electropherograms (Figure 2B and zoom-in B1 and B2). These &#34;extra&#34; peaks could explain the lower RIN values obtained.
The equipment also calculates sample concentrations considering the standard ladder in lane 1 as a reference. As expected, the TOTAL samples present higher concentration values than the PAR samples. Still, even for these, there is enough RNA to proceed with library preparation and RNA-seq since sample quantity requirements typically recommend at least 1.0 &#181;g.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclavable solutions to be stored at room temperature</td>
		</tr>
		<tr>
			<td>2 M Tris-HCl pH 9.0</td>
		</tr>
		<tr>
			<td>2 M KCl&#160;</td>
		</tr>
		<tr>
			<td>0.5 M EGTA pH 8.0 (adjusted with 10 M NaOH)</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
		</tr>
		<tr>
			<td>20% (v/v) PTE (shake bottle before pipetting the solution)</td>
		</tr>
		<tr>
			<td>10% DOC&#160;</td>
		</tr>
		<tr>
			<td>20% Detergent mix: 20% (w/v) Brij L23, 20% (v/v) Trit&#243;n X-100, Igepal CA 360, 20% (v/v) Tween 20 (dissolve while heating at 60 &#176;C)</td>
		</tr>
		<tr>
			<td>Filter-sterilizable solutions to be frozen at -20 &#176;C</td>
		</tr>
		<tr>
			<td>0.5 M DTT</td>
		</tr>
		<tr>
			<td>0.5 M PMSF</td>
		</tr>
		<tr>
			<td>50 mg.mL-1 cycloheximide (CHX) (in EtOH)</td>
		</tr>
		<tr>
			<td>50 mg.mL-1 chloranphenicol (CHL) (in EtOH)</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock solution</td>
			<td>Volume (&#181;L) for 10 mL PEB</td>
			<td>Volume (&#181;L) for 2 mL RB</td>
		</tr>
		<tr>
			<td>2 M Tris, pH 9.0</td>
			<td>1,000</td>
			<td>200</td>
		</tr>
		<tr>
			<td>2 M KCl</td>
			<td>1,000</td>
			<td>200</td>
		</tr>
		<tr>
			<td>0.5 M EGTA pH 8.0</td>
			<td>500</td>
			<td>100</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>356</td>
			<td>70</td>
		</tr>
		<tr>
			<td>20% (v/v) PTE</td>
			<td>500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>10% DOC</td>
			<td>1,000</td>
			<td>-</td>
		</tr>
		<tr>
			<td>20% Detergent mix</td>
			<td>500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>0.5 M DTT</td>
			<td>100</td>
			<td>20</td>
		</tr>
		<tr>
			<td>0.5 M PMSF</td>
			<td>20</td>
			<td>-</td>
		</tr>
		<tr>
			<td>50 mg.mL-1 CHX</td>
			<td>10</td>
			<td>2</td>
		</tr>
		<tr>
			<td>50 mg.mL-1 CHL</td>
			<td>10</td>
			<td>2</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>5,004</td>
			<td>1,406</td>
		</tr>
		<tr>
			<td colspan="3">2 M sucrose solution (68.5%): 68.5 g of sucrose in water. Keep at room temperature (RT).&#160;</td>
		</tr>
		<tr>
			<td colspan="3">10x salt solution: 0.4 M Tris-HCl pH 8.4, 0.2 M KCl, 0.1 M MgCl2; autoclave for 15 min, freeze at -20 &#176;C.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers and solutions used in this protocol. The volumes given are for six samples. Abbreviations: EGTA = ethylene glycol-bis(2-aminoethylether)-N,N,N&#180;,N&#180;-tetraacetic acid; PTE = polyoxyethylene (10) tridecyl ether; DOC = sodium deoxycholate; DTT = dithiothreitol; PMSF = phenylmethanesulfonyl fluoride.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose layer (%)</td>
			<td>Sucrose 2 M (mL)</td>
			<td>Salt solution 10x (mL)</td>
			<td>CHX 50 mg.mL-1 (&#181;L)</td>
			<td>CHL 50 mg.mL-1 (&#181;L)</td>
			<td>H2O (mL)</td>
		</tr>
		<tr>
			<td>12</td>
			<td>5.25</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
			<td>21.75</td>
		</tr>
		<tr>
			<td>33.5</td>
			<td>14.7</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
			<td>12.3</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of the two sucrose layers (12% and 33.5%). The volumes given are for the preparation of six cushions.

Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Polysome Purification from Soybean Symbiotic Nodules

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

polysome-purification-from-soybean-symbiotic-nodules

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.4K Views.  Universidad de la República. Dit protocol is belangrijk omdat het, voor zover wij weten, het eerste is dat een nieuw op centrifuge gebaseerd metaal beschrijft voor kinetische polysoomzuivering van sojasymbiotische knobbeltjes. Het grote voordeel van deze techniek is dat in combinatie met RNA-Seq, de studie van de relatie mogelijk is en georganiseerd in een complex weefsel zoals de symbiotische knobbel. Weeg om te beginnen ongeveer 0,2 gram intacte knobbeltjes in een weegschaal en breng ze over in een voorgekoelde buis va...

Video: Polysome Purification from Soybean Symbiotic Nodules

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Chemistry

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunology and Infection

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Biology

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. ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

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 ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Polysome Purification from Soybean Symbiotic Nodules

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