The authors thank Ching Lee for help with audio recording.The research was supported by the Start-up funds from Texas Tech University Health Sciences Center and by the Center of Excellence for Translational Neuroscience and Therapeutics (CTNT) grant PN-CTNT 2017-05 AKHRJDHW to A.L.K.; in part by NIH grant R01AI099380 to K.Z. James C. Huffman and Kristen R. Baca were CISER (Center for the Integration of STEM Education &#38; Research) scholars and were supported by the program.

All animal treatments and handling of tissues obtained in the study were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Science Center in accordance with the National Institutes of Health animal welfare guidelines, protocol number 96005. Please sacrifice vertebrate animals and prepare tissues according to the guidelines from the Institutional Animal Care and Use Committee. If lacking such a committee, please refer to the National Institutes of Health animal welfare guidelines. Adult (&#62;60 day old) C57BL/6 mice were used. All animals and tissues were obtained according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Sciences Center in accordance with the National Institutes of Health animal welfare guidelines. For euthanasia, a single mouse was placed in a small chamber, and the air was displaced gradually with about 30% carbon dioxide to anesthetize and minimize the distress of the animal. Following cessation of breathing, we used cervical dislocation to confirm the death of the animal before harvesting tissues.
Caution: All work with live Leishmania and cultured human cells was done in biosafety cabinet in BSL-2 certified laboratory.
1. Preparation of Cytoplasmic Lysates from Leishmania Major , Cultured Human Cells, and Mouse Tissues
NOTE: There are several differences in the lysate preparations from the different source materials. Other steps including sucrose gradient preparation and polysomal fractionation are identical and do not depend on sample source.
<ol>
	<li>Leishmania major cytoplasmic lysate preparation
	<ol>
		<li>Inoculate Leishmania major (FV1 strain) cells in 30 mL of 1x M199 medium29 containing 10% Fetal Bovine Serum (FBS) and penicillin/streptomycin mixture (100 units and 100 &#956;g/mL correspondingly) at density of 1x105 cells/mL. 
		NOTE: All steps involving Leishmania major cells must be conducted in a biosafety cabinet.</li>
		<li>Place cells in the incubator and grow them at 27 &#176;C until the logarithmic phase (mid log corresponds to 5x106 cells/mL). It usually takes about two days to grow.</li>
		<li>Add cycloheximide to Leishmania major culture to a final concentration of 100 &#956;g/mL to arrest the ribosomes on translated mRNAs. Place cells back in the incubator for 10 min at 27 &#176;C.</li>
		<li>After cycloheximide treatment is completed, transfer cells to a 50 mL conical tube and spin them at 1,800 x g and 4 &#176;C for 8 min. Discard supernatant.</li>
		<li>Wash cells with 30 mL of Dulbecco&#39;s phosphate buffered saline (DPBS). Centrifuge at 1,800 x g and 4 &#176;C for 8 min.</li>
		<li>Discard the supernatant. Resuspend cells in 1 mL of DPBS.</li>
		<li>Take an aliquot of cells and mix it with 3.5% formaldehyde solution.</li>
		<li>Count cells by hemocytometer and determine their concentration. Transfer the desired number of cells into microfuge tube. Lysate prepared from 0.5x108-2x108 cells/mL is sufficient for one sucrose gradient loading.</li>
		<li>Spin the cells at 1,800 x g and 4 &#176;C for 8 min. Discard the supernatant.</li>
		<li>Resuspend the cell pellet on ice in 1 mL of lysis buffer containing protease inhibitors and RNase inhibitor (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 mM MgCl2, 2 mM DTT, 1% NP-40, 1x protease inhibitor cocktail&#160;(EDTA-free), 200 units/mL RNase inhibitor).</li>
		<li>Pass the lysate through a 23-gauge needle three times. The lysate should become transparent after passage through the needle.</li>
		<li>Centrifuge at 11,200 x g and 4 &#176;C for 10 min to clarify lysate. Transfer the clarified lysate to a fresh tube and keep it on ice until sucrose gradient ultracentrifugation.</li>
		<li>Collect 400-500 &#956;L of the lysate as input (to analyze later), freeze it right away in liquid nitrogen for future protein analysis or add RNA purification reagent before freezing for RNA analysis.</li>
	</ol>
	</li>
	<li>Cytoplasmic lysate preparation from cultured human HeLa cells
	<ol>
		<li>Split HeLa cells and seed them into 20 mL of the DMEM medium containing 10% FBS and penicillin/streptomycin mixture (100 units and 100 &#956;g/mL correspondingly) with cell count 2x105 cells/mL in a 15 cm plate.</li>
		<li>Grow HeLa cells at 37 &#176;C, 5% CO2 for 20-24 h. Perform plasmid DNA transfection according to manufacturer&#39;s protocols.</li>
		<li>Propagate cells for 24 h after transfection at 37 &#176;C, 5% CO2.</li>
		<li>Add cycloheximide to grown HeLa cells to the final concentration of 100 &#956;g/mL to arrest the ribosomes on translated mRNAs and incubate cells for 10 min at 37 &#176;C, 5% CO2. Aspirate medium. Wash the cells twice with cold DPBS on ice.</li>
		<li>Add 500 &#956;L of lysis buffer (20 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 200 units/mL of RNase inhibitor or 1 mg/mL heparin) to the plate and scrape the cells on ice.</li>
		<li>Transfer the lysed cells to the microfuge tube. Adjust the concentration of NP-40 to 0.5% and MgCl2 to 5 mM according to the increased volume of the sample.</li>
		<li>Pass the lysate through a 23-gauge needle 3-6 times.</li>
		<li>Spin at 11,200 x g and 4 &#176;C for 8 min to clarify lysate. After centrifugation, transfer supernatant to a new tube. Use a spectrophotometer to evaluate cell lysis efficiency and to determine sample amount for the loading on the gradient. Add 10 &#956;L of sample to 0.5 mL of 0.1% Sodium Dodecyl Sulfate (SDS). Blank against 0.1% SDS. Measure absorbance at 260 nm. Expected absorbance value is around 15-20 units/mL.</li>
		<li>Dilute all samples with lysis buffer to the same absorbance value before sucrose gradient centrifugation. Keep samples on ice until sucrose gradient centrifugation.</li>
	</ol>
	</li>
	<li>Cytoplasmic lysate preparation from mouse testis
	<ol>
		<li>Dissect the mouse testis. Make a small incision in the tunica albuginea and collect the seminiferous tubules of the testes and transfer them in a 15 mL conical tube containing 5 mL of DPBS supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF).</li>
		<li>Mix the tissue vigorously by inverting several times. Allow the tissue to settle at unit gravity on ice for 5 min.</li>
		<li>Remove and discard the cloudy buffer containing connective cells and tissue fragments. Repeat the procedure 2-3 more times. The remaining white pellet is enriched for seminiferous tubules and germ cells.</li>
		<li>Transfer the seminiferous tubule pellet to a 2 mL microcentrifuge tube and spin at 500 x g for 1 min. Discard the supernatant.</li>
		<li>Add 500 &#181;L of lysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 1 mg/mL heparin or 200 units/mL of RNase inhibitor) to the tubules. Use a pipette to triturate the tissue.</li>
		<li>Transfer the suspension to a small (0.5-1.0 mL) Dounce homogenizer. Disrupt the tissue with seven to eight strokes of the glass pestle.</li>
		<li>Transfer the lysate to a 1.5 mL microcentrifuge tube.</li>
		<li>Centrifuge the sample at 12,000 x g and 4 &#176;C for 8 min to clear the lysate. Transfer the supernatant to a new tube and store on ice until loading on the sucrose gradient.</li>
		<li>Collect 50 &#956;L of the lysate as input sample, freeze it right away at -80 &#176;C for future protein analysis; or add RNA purification reagent before freezing for RNA analysis.</li>
	</ol>
	</li>
</ol>
2. Sucrose Gradient Preparation and Ultracentrifugation
<ol>
	<li>Prepare two sucrose gradient solutions (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail), containing either 10% sucrose or 50% sucrose. (Tris-HCl, pH 7.4, can be used instead of HEPES). Add 200 units/mL RNase inhibitor or 1 mg/mL heparin according to the experimental design. Place an ultracentrifuge tube for SW 41 rotor into the marker block and draw the line along the upper level of the block. Transfer the tube into a stable rack.</li>
	<li>Take a 10-mL syringe with the layering device attached and fill the syringe with 10% sucrose solution (prepared as above). Gently release it at the bottom of the ultracentrifuge tube until it reaches the mark on the tube.</li>
	<li>Fill another syringe with 50% sucrose solution and carefully insert its layering device through the 10% sucrose layer to the bottom of the tube. Gently release sucrose solution starting from the bottom until it reaches the mark on the tube. Seal the tube with the provided cap.</li>
	<li>To prepare the sucrose gradient, turn the gradient maker device ON. Level the plate using the UP or DOWN buttons and press DONE. Leveling is important for linearity of the gradient.</li>
	<li>After leveling the plate press GRAD to open the gradient menu. Go to LIST on the gradient menu and select the SW 41 Ti rotor. Then choose the desired sucrose gradient from the list of the menu using UP and DOWN buttons. Press USE.</li>
	<li>Place the gradient tube holder on the gradient maker plate. Transfer the tube into the holder. Up to 6 gradients can be prepared at the same time. Press RUN. The gradient maker rotates the tubes at the programmed speed and angles forming a linear gradient. It will take only a few minutes to prepare the gradient.</li>
	<li>When process is completed, place the tubes in a rack. Take the caps off. Remove the same volume as the sample volume from the top of the ultracentrifuge tubes.</li>
	<li>Carefully load 400-500 &#956;L of lysate containing 15-20 A260 units of polysomes on the top. Place the tubes in the rotor buckets and balance them.</li>
	<li>Centrifuge at 260,000 x g and 4 &#176;C for 2 h using SW 41 rotor.</li>
</ol>
3. Polysome Fractionation and Sample Collection
NOTE: While lysate preparations have some differences depending on the source, gradient preparation and polysome fractionation protocols are the same for all types of lysates.
<ol>
	<li>After completion of the ultracentrifugation, place the rotor buckets with the tubes on ice. Turn fraction collector and gradient fractionator ON. Click SCAN on the fractionator menu. Put a rack with 24 collection tubes into the fraction collector.</li>
	<li>Fill up a rinse reservoir on the side of the fractionator with deionized water. Press the RINSE key for 10 s to rinse the pump on fractionator. Attach a rinse adaptor with the syringe filled with water to the piston for the calibration.</li>
	<li>Open the fractionator software on the computer. Press CALIBRATE. Use the DEFAULT settings and press OK. Be ready to inject water from syringe.</li>
	<li>Press OK to do calibration. Immediately start injecting water for the next 5 s. During this time, water will flow through the UV detector flow cell and the instrument will be calibrated. The sign ZERO CALIBRATION COMPLETED will appear. The instrument is ready for fractionation.</li>
	<li>Remove the rinse adaptor with the syringe. Attach a tip to the piston of the fractionator.</li>
	<li>Open the brass air valve and press AIR key for 10 s to dry tubing and flow cell. Close the air valve.</li>
	<li>Gently remove the gradient tube from the rotor bucket and place it in the rack. Apply the tube holder cap to the top of the tube and carefully move the tube into the tube holder and lock it in position.</li>
	<li>Place the holder under the piston of the fractionator. Often, polysomal bands can be seen by eye. Introduce the desired settings for fraction numbers and volume (24 fractions at 500 &#956;L/fraction are usually sufficient). Name the file appropriately. Press OK, and then GO TO GRAPH button. In the next window, press START SCAN. Settings will appear, press OK. The collector will move from the gutter to the first fraction and the piston will move into the tube. When the piston reaches the top of the gradient it will slow to the selected speed and the fractions will be collected. When completed, the piston moves out of the gradient tube.</li>
	<li>Open the brass air valve and press AIR key on the fractionator to retrieve the last fraction.</li>
	<li>Move the tubes from the rack on ice.</li>
	<li>Add 2 volumes of RNA purification reagent to each fraction and flash freeze in liquid nitrogen until RNA purification. Alternatively, if protein needs to be analyzed, add trichloroacetic acid to the final concentration of 10% to concentrate them for Western blotting (see Section 8).</li>
</ol>
4. Preparation of Synthetic RNA In Vitro for Normalization of mRNAs Levels During RT-qPCR Data Analysis
NOTE: The E. coli OmpA mRNA is used in this protocol for normalization. Any other RNA that does not have extensive identity with the mRNAs of the studied organism (mammalian or Leishmania) can be used.
<ol>
	<li>Prepare the OmpA DNA fragment containing SP6 promoter sequence by a standard PCR reaction from a plasmid containing OmpA gene30.</li>
	<li>Prepare 100 &#181;L of the mixture: 80 mM HEPES-KOH, pH 7.5, 16 mM MgCl2, 2 mM Spermidine, 10 mM DTT, 3 mM ATP, 3 mM CTP, 3 mM UTP, 3 mM GTP, 0.5 U/&#956;L RNase inhibitor, 1 &#956;g of OmpA PCR DNA, 3 &#956;L SP6 RNA polymerase, 0.005 U/&#956;L pyrophosphatase.</li>
	<li>Incubate at 40 &#176;C for 2 h.</li>
	<li>Purify RNA by a RNA purification kit.</li>
	<li>Measure concentration by spectrophotometer and examine by agarose gel electrophoresis.</li>
</ol>
5. RNA Isolation from Gradient Fractions and cDNA Preparation
NOTE: Proceed directly with this protocol for RNA purification if an RNase inhibitor was used as a ribonuclease inhibitor. However, when used as a ribonuclease inhibitor, heparin will inhibit reverse transcriptase used in cDNA preparation. Therefore, additional purification of RNA will be needed if heparin was used in the lysis buffer and gradient. See Section 6 to prepare RNA for cDNA synthesis if heparin was used.
<ol>
	<li>Thaw the samples containing RNA purification reagent, add 20 ng of synthetic RNA as internal control for normalization of RT-qPCR results. Proceed with RNA preparation according to the manufacturer&#39;s protocol except one modification. Add 1 &#181;L of RNA grade glycogen (20 &#181;g) prior isopropanol precipitation. Dissolve RNA pellets in 20-25 &#181;L of RNase-free water. 
	NOTE: Glycogen serves as a carrier and helps to avoid losses and visualize RNA pellet during purification. OmpA mRNA is used for further normalization in RT-qPCR reactions.</li>
	<li>Measure RNA concentration using spectrophotometer to ensure adequate yield. Combine equal volumes of RNA fractions containing 40S, 60S and monosomes as prepolysomes. Fractions containing 2-4 ribosomes combine as light polysomes and fractions with 5-8 ribosomes combine as heavy polysomes.</li>
	<li>Use 5-10 &#181;L of RNA from combined fractions to prepare cDNAs using a kit and following manufacturer&#39;s recommendations.</li>
	<li>Add 80 &#181;L of nuclease free water to 20 &#181;L of cDNA. Freeze the cDNA samples at -20 &#176;C.</li>
</ol>
6. RNA Purification from Heparin Contamination
NOTE: Heparin inhibits nucleic acid processing enzymes such as reverse transcriptase. Therefore, use this additional purification protocol when heparin is used in the lysis buffer and/or in the gradient.
<ol>
	<li>Add LiCl to a 1 M final concentration to the purified RNA samples.</li>
	<li>Mix the samples and incubate on ice for 1 h.</li>
	<li>Spin the samples at 16,000 x g and 4 &#176;C for 15 min.</li>
	<li>Remove the supernatant as complete as possible using a pipette.</li>
	<li>Air-dry the pellets for about 5 min.</li>
	<li>Re-suspend the pellets in the initial volume of RNase-free water.</li>
	<li>Perform spectrophotometric measurement at 260 nm to determine the concentration of the RNA. Usually, the loss of the sample is minimal.</li>
</ol>
7. RT-qPCR and Data Analysis of mRNA Distribution
<ol>
	<li>Combine 10.2 &#956;L of water, 20 &#956;L of SYBR Green, 4.8 &#956;L of gene specific primers (2.5 &#956;M each set), 5 &#956;L of cDNA, mix well and load 10 &#956;L per well in triplicates into 384-well plate.</li>
	<li>Cover plate with adhesive film tightly and centrifuge plate at 1,800 x g for 5 min.</li>
	<li>Using a Real-Time PCR instrument set up qPCR reaction under conditions shown in the Table 1.</li>
	<li>Using the cycle threshold (CT) values and the comparative CT (&#916;&#916;CT) method31 calculate the percentage (%) of mRNA distribution in prepolysomes, light, and heavy polysomes as described32 with one modification. Use synthetic RNA (OmpA here) for data normalization in RT-qPCR data analysis. The synthetic RNA provides a normalization control that allows to calculate relative mRNAs levels and compare them in different fractions of a gradient.</li>
</ol>
8. Analysis of Proteins in Polysomal Fractions by Western Blotting
<ol>
	<li>From a 100% (w/v) stock, add trichloroacetic acid (TCA) to the selected fractions (500 &#956;L) to a final concentration of 10%, keep on ice for at least 15 min, centrifuge in a microfuge for 5 min, discard supernatant, wash twice with ice-cold acetone and dissolve in 25 &#956;L of SDS-PAGE sample loading buffer for electrophoresis.</li>
	<li>Load on the SDS-PAGE and conduct standard electrophoresis with following transfer to the PVDF membrane. Proceed to Western blotting33.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+differentiation+in+Leishmania+lifecycle+progression:+post-transcriptional+control+conducts+the+orchestra.">Developmental differentiation in Leishmania lifecycle progression: post-transcriptional control conducts the orchestra.</a> Current Opinions in Microbiology. 34, 82-89 (2016).</li><li>Soto, M., 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=Cell-cycle-dependent+translation+of+histone+mRNAs+is+the+key+control+point+for+regulation+of+histone+biosynthesis+in+Leishmania+infantum.">Cell-cycle-dependent translation of histone mRNAs is the key control point for regulation of histone biosynthesis in Leishmania infantum.</a> Biochemical Journal. 379, 617-625 (2004).</li><li>McNicoll, F., 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=Distinct+3+'-untranslated+region+elements+regulate+stage-specific+mRNA+accumulation+and+translation+in+Leishmania.">Distinct 3 '-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania.</a> Journal of Biological Chemistry. 280 (42), 35238-35246 (2005).</li><li>Folgueira, C., 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=The+translational+efficiencies+of+the+two+Leishmania+infantum+HSP70+mRNAs,+differing+in+their+3+'-untranslated+regions,+are+affected+by+shifts+in+the+temperature+of+growth+through+different+mechanisms.">The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3 '-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.</a> Journal of Biological Chemistry. 280 (42), 35172-35183 (2005).</li><li>Dumas, C., Chow, C., Muller, M., Papadopoulou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+class+of+developmentally+regulated+noncoding+RNAs+in+Leishmania.">A novel class of developmentally regulated noncoding RNAs in Leishmania.</a> Eukaryotic Cell. 5 (12), 2033-2046 (2006).</li><li>Kapler, G. M., Coburn, C. M., Beverley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+the+human+parasite+Leishmania+major+delineates+a+30-kilobase+region+sufficient+for+extrachromosomal+replication+and+expression.">Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.</a> Molecular Cell Biology. 10 (3), 1084-1094 (1990).</li><li>Karamyshev, A. L., Johnson, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+SecA+association+with+signal+sequences+in+ribosome-bound+nascent+chains:+a+potential+role+for+SecA+in+ribosome+targeting+to+the+bacterial+membrane.">Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.</a> Journal of Biological Chemistry. 280 (45), 37930-37940 (2005).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nature Protocols. 3 (6), 1101-1108 (2008).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Fractionation+to+Analyze+mRNA+Distribution+Profiles.">Polysome Fractionation to Analyze mRNA Distribution Profiles.</a> Bio Protocols. 7 (3), (2017).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (1989).</li><li>Patrick, A. E., Karamyshev, A. L., Millen, L., Thomas, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+CFTR+transmembrane+span+integration+by+disease-causing+mutations.">Alteration of CFTR transmembrane span integration by disease-causing mutations.</a> Molecular Biology of the Cell. 22 (23), 4461-4471 (2011).</li><li>Kleizen, B., van Vlijmen, T., de Jonge, H. R., Braakman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+CFTR+is+predominantly+cotranslational.">Folding of CFTR is predominantly cotranslational.</a> Molecular Cell. 20 (2), 277-287 (2005).</li><li>van den Elzen, A. M., Schuller, A., Green, R., Seraphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dom34-Hbs1+mediated+dissociation+of+inactive+80S+ribosomes+promotes+restart+of+translation+after+stress.">Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress.</a> EMBO Journal. 33 (3), 265-276 (2014).</li><li>Morita, M., 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=mTOR+Controls+Mitochondrial+Dynamics+and+Cell+Survival+via+MTFP1.">mTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1.</a> Molecular Cell. 67 (6), 922-935 (2017).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li></ol>

The authors have nothing to disclose.

Polysome fractionation by sucrose gradient combined with RNA and protein analysis of fractions is a powerful method to analyze translational status of individual mRNAs or the whole translatome as well as roles of protein factors regulating translational machinery during normal physiological or disease state. Polysomal profiling is an especially suitable technique to study translational regulation in organisms such as trypanosomatids including Leishmania where transcriptional control is largely absent and gene ex...

Regulation of gene expression in cells is controlled by transcriptional, posttranscriptional and posttranslational mechanisms. Advances in deep RNA sequencing allow the study of steady-state mRNA levels on a genome-wide scale at an unprecedented level. However, recent findings revealed that steady-state mRNA level does not always correlate with protein production1,2. The fate of an individual transcript is very complex and depends on many factors like internal/external stimuli, stress, etc. Regulation of gene expression during protein synthesis provides another layer of expression control necessary for a rapid response in changing conditions. Polysome (or &#34;polyribosome&#34;) profiling, the separation and visualization of actively translating ribosomes, is a powerful method to study the regulation of protein synthesis. Although, its first experimental applications appeared in the 1960s3, polysome profiling is currently one of the most important techniques in protein translation studies4. Single mRNAs can be translated by more than one ribosome leading to the formation of a polysome. Transcripts can be stalled on ribosomes with cycloheximide5 and mRNAs containing different numbers of polysomes can be separated in the process of polysome fractionation by sucrose gradient ultracentrifugation6,7,8,9. RNA analysis of polysomal fractions then allows measurement of changes in the translational states of individual mRNAs on genome-wide scale and during different physiological conditions4,7,10. The method has been also used to reveal the roles of 5&#39;UTR and 3&#39;UTR sequences in control of mRNA translatability11, examine the role of miRNAs in translational repression12, uncover defects in ribosome biogenesis13, and understand the role of ribosome-associated proteins with human diseases14,15. During the last decade, a growing role for regulation of gene expression during translation has emerged that illustrates its importance in human diseases. The evidence for translational control in cancer, metabolic and neurodegenerative diseases is overwhelming15,16,17,18. For example, dysregulation of eIF4E-dependent translational control contributes to autism related deficits15 and FMRP is involved in stalling of ribosomes on mRNAs linked to autism14. Thus, polysomal profiling is a very important tool to study defects in translational regulation in multiple human diseases.
Protein analysis of polysomal fractions under different physiological conditions dissects the function of factors associated with ribosomes during translation. The polysome profiling technique has been used in many species including yeast, mammalian cells, plants, and protozoa10,19,20,21. Protozoan parasites like Trypanosoma and Leishmania exhibit limited transcriptional control of gene expression. Their genomes are organized into polycistronic gene clusters that lack promoter-regulated transcription22. Instead, developmental gene expression is predominantly controlled at the level of protein translation and mRNA stability in trypanosomatid species23,24. Therefore, understanding of translational control in the absence of transcriptional regulation is particularly important for these organisms. Polysomal profiling is a powerful tool to study posttranscriptional regulation of gene expression in Leishmania25,26,27,28.
The recent progress in detection of individual mRNAs levels by real time quantitative PCR (RT-qPCR) and full transcriptome by next-generation sequencing, as well as proteomics technologies, brings resolution and advantages of polysomal profiling to a new level. The use of these methods can be further extended by analysis of individual polysomal fractions by deep RNA sequencing combined with proteomic analysis to monitor the translational status of cells on a genome-wide scale. This allows the identification of new molecular players regulating translation under different physiological and pathological conditions. Here, we present a universal polysome profiling protocol used on three different models: the parasite Leishmania major, cultured human cells, and animal tissues. We present advice on the preparation of cell lysates from different organisms, optimization of gradient conditions, choice of RNase inhibitors and application of RT-qPCR, Western blot and RNA electrophoresis to analyze polysome fractions in this study.

<table><tbody><tr><td>&lt;strong&gt;Instruments:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gradient master</td><td>Biocomp Instruments Inc.</td><td>108</td><td></td></tr><tr><td>Piston Gradient Fractionator</td><td>Biocomp Instruments Inc.</td><td>152</td><td></td></tr><tr><td>Fraction collector</td><td>Gilson, Inc.</td><td>FC203B</td><td></td></tr><tr><td>NanoDrop One</td><td>Thermo Scientific</td><td>NanoDrop One</td><td></td></tr><tr><td>Nikon inverted microscope</td><td>Nikon</td><td>ECLIPSE Ts2-FL/Ts2</td><td></td></tr><tr><td>2720 Thermal Cycler</td><td>Applied Biosystems by Life Technologies</td><td>4359659</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Panasonic Healthcare Co.</td><td>MCO-170A1CUV</td><td></td></tr><tr><td>HERATHERM incubator</td><td>Thermo Scientific</td><td>51028063</td><td></td></tr><tr><td>Biological Safety Cabinet, class II, type A2</td><td>NuAire Inc.</td><td>NU-543-400</td><td></td></tr><tr><td>Revco freezer</td><td>Revco Technologies</td><td>ULT1386-5-D35</td><td></td></tr><tr><td>Beckman L8-M Ultracentifuge</td><td>Beckman Coulter</td><td>L8M-70</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW41</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Swing-bucket rotor</td><td>Eppendorf</td><td>A-4-62</td><td></td></tr><tr><td>Fixed angle rotor</td><td>Eppendorf</td><td>F-45-30-11</td><td></td></tr><tr><td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td><td>Applied Biosystems by life technologies</td><td>4470661</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>02-671-51B</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Triax software&amp;nbsp;</td><td>Biocomp Instruments Inc.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Counting slides, dual chamber for cell counter</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td><td>Seton Scientific</td><td>7030</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>BD</td><td>309646</td><td></td></tr><tr><td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td><td>BD</td><td>10020439</td><td></td></tr><tr><td>Nunclon Delta Surface plate, 14 cm</td><td>Thermo Scientific</td><td>168381</td><td></td></tr><tr><td>Nunclon Delta Surface plate, 9 cm</td><td>Thermo Scientific</td><td>172931</td><td></td></tr><tr><td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td><td>Thermo Scientific</td><td>569-0020</td><td></td></tr><tr><td>BioLite 75 cm&lt;sup&gt;3&lt;/sup&gt; flasks</td><td>Thermo Scientific</td><td>130193</td><td></td></tr><tr><td>Nunc 50 mL conical centrifuge tubes</td><td>Thermo Scientific</td><td>339653</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trizol LS</td><td>Ambion by Life Technologies</td><td>10296028</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1378-5KG</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium-high glucose (DMEM)</td><td>Sigma</td><td>D6429-500ML</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F0926-50ML</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S)</td><td>Sigma</td><td>P0781-100ML</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Sigma</td><td>D8537-500ML</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td><td>Acros Organics</td><td>AC413415000</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma</td><td>P9541-500G</td><td></td></tr><tr><td>Nonidet P 40 (NP-40)</td><td>Fluka (Sigma-Aldrich)</td><td>74385</td><td></td></tr><tr><td>Recombinant Rnasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2511</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3993-1MU</td><td></td></tr><tr><td>cOmplete Mini EDTA-free protease inhibitors</td><td>Roche Diagnostics</td><td>11836170001</td><td></td></tr><tr><td>Glycogen</td><td>Thermo Scientific</td><td>R0551</td><td></td></tr><tr><td>Water</td><td>Sigma</td><td>W4502-1L</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698-1G</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>194002</td><td></td></tr><tr><td>Dithiotreitol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Fisher Scientific</td><td>BP-1302-10</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td><td>Fisher Scientific</td><td>S316-212</td><td></td></tr><tr><td>Optimem</td><td>Life Technologies</td><td>22600050</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Sigma</td><td>P8833-100MG</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3KG</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T4049-100ML</td><td></td></tr><tr><td>Hgh Capacity cDNA Reverse Transcriptase Kit</td><td>Applied Biosystems by life technologies</td><td>4368814</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>Applied Biosystems by life technologies</td><td>4367659</td><td></td></tr><tr><td>HCl</td><td>Fisher Scientific</td><td>A144SI-212</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP26324</td><td></td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Sigma</td><td>221473-500G</td><td></td></tr><tr><td>Anti-RPL11 antibody</td><td>Abcam</td><td>ab79352</td><td></td></tr><tr><td>Ribosomal protein S6 (C-8) antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-74459</td><td></td></tr><tr><td>1xM199</td><td>Sigma</td><td>M0393-10X1L</td><td></td></tr><tr><td>Lithium cloride</td><td>Sigma</td><td>L-9650</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Gel Loading Buffer II</td><td>Thermo Scientific</td><td>AM8546G</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Thermo Scientific</td><td>16500-100</td><td></td></tr><tr><td>Trichloracetic acid (TCA)</td><td>Fisher Scientific</td><td>A322-100</td><td></td></tr><tr><td>SuperSignal West Pico PLUS chemiluminescent substrate</td><td>Thermo Scientific</td><td>34580</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific</td><td>BP531-500</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma</td><td>L5750-1KG</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma</td><td>P7626-5G</td><td></td></tr><tr><td>RNeasy Mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate (ATP)</td><td>Sigma</td><td>A1852-1VL</td><td></td></tr><tr><td>Cytosine 5&#039;-triphosphate disodium salt hydrate (CTP)</td><td>Sigma</td><td>C1506-250MG</td><td></td></tr><tr><td>Uridine 5&#039;-triphosphate trisodium salt hydrate (UTP)</td><td>Sigma</td><td>U6625-100MG</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate (GTP)</td><td>Sigma</td><td>G8877-250MG</td><td></td></tr><tr><td>SP6 RNA Polymerase</td><td>NEB</td><td>M0207S</td><td></td></tr><tr><td>Pyrophoshatase</td><td>Sigma</td><td>I1643-500UN</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S0266-1G</td><td></td></tr></tbody></table>

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.

In this study, we describe the application of the polysomal profiling technique to three different sources: parasitic Leishmania major, cultured human cells, and mouse testis. Leishmania cells freely grow in the liquid media in suspension, cultured human cells grow in the adherent monolayer on plates, and the mouse testis represents a tissue sample. The method can be easily adjusted to other types of freely grown cells in suspension, different types of tissues, or from a...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

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.

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 ...

polysome-profiling-in-leishmania-human-cells-and-mouse-testis

Research

JoVE Journal

Biochemistry

18.0K Views.  Texas Tech University Health Sciences Center. Het algemene doel van Polysoom profiling techniek is analyse van translationeel activiteiten van individuele mRNAs of transcriptome mRNAs tijdens de eiwitsynthese. De methode is belangrijk voor de studie van eiwit synthese verordening, vertaling activering en onderdrukking in gezondheid en meerdere ziekten bij de mens.

Video: Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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 ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Immunology and Infection

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

Genetics

Polysome Profiling without Gradient Makers or Fractionation Systems

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

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

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

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

Developmental Biology

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 ...

Neuroscience

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

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

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

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

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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