Polisoma Profiling nel testicolo di topo, cellule umane e di Leishmania

The overall goal of this technique is to study engagement of indivdual mRNA's with polysomes during translation. Polysome profiling is a powerful technology to study mRNA engagement with ribosomes and polysomes. The technique is important for studies of protein synthesis regulation, translation activation, and repression.

Here our team presents the techniques for polysomal fractionation, and analysis of fractions on the example of three organisms. Parasite Leishmania major, cultured human cells, and an animal tissue. This approach consists of four major steps, lysate preparation, sucrose gradient preparation and ultracentrifugation, polysome fractionation, and sample collection.

Analysis of the fractions. Cells from different sources are collected, washed and lysed in lysis buffer by passage through a needle or Dounce homogenizer. Centrifugation is used to remove cell debris clarifying the lysate.

A continuous sucrose gradient is formed by the mixing of 10%and 50%sucrose solutions in a gradient maker. Lysate is loaded on the top of the gradient. Ultracentrifugation separates mrNA's associated with different number of ribosomes.

Which is monitored by a UV Detector during fractionation, forming distinct absorbant spectrums. Lysate preparation. There are some differences in the preparation of the lysate depending on the source.

After lysate preparation, procedures are identical regardless of the sample origin. Leishmania major Cytoplasmic Lysate preparation. Place Leishmania major cells in 30 milliliter of medium at the density of 100, 000 cells per milliliter.

This step is conducted in a biosafety cabinet. Place the flask with Leishmania culture in the incubator and grow cells at 27 degrees Celsius, until the logarithmic phase. It usually takes about two days to grow.

Leishmania major cells are easily visible under the microscope. Add Cycloheximide to the grown Leishmania culture to a final concentration of 100 micrograms per milliliter, to arrest ribosomes on translated mRNAs. Place cells back in the incubator for ten minutes at 27 degrees Celsius.

After cycloheximide treatment is completed transfer cells to a 50 milliliter conical tube and spin it 1, 800 g for 8 minutes. Discard supernatant. Wash cells with 30 milliliter of pbs and centrifuge.

Discard the supernatant and re-suspend the cells in one milliliter of pbs. Take an aliquot of cells and mix it with the formaldehyde solution. Then count cells by hemocytometer.

Transfer the desired number of cells into microfuge tube. Spin the cells at 1, 800 g for 8 minutes at four degrees Celsius. Then discard the supernatant.

Re-suspend the cell pellet in one milliliter of lysis buffer containing protease inhibitors and RNAsin. Pass the cells through a 23 gauge needle three times. After passing through the needle, the lysate becomes transparent.

Centrifuge at 11, 200 g for ten minutes at four degrees Celsius to clarify lysate. Transfer the clarified lysate to a fresh microfuge tube and keep it on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from cultured human cells.

Grow HeLa cells in a large plate in the incubator at 37 degrees Celsius, with 5%CO2. Add cycloheximide to grown HeLa cells to the final concentration of 100 micrograms per milliliter to arrest the ribosomes on translated mRNA's. Incubate cells for ten minutes at 37 degrees Celsius with 5%CO2.

Aspirate medium and wash plate on ice twice with cold pbs. Add 500 microliters of lysis buffer to the plate and scrape the cells. Transfer the lysed cells to the microfuge tube.

Pass the cells through a 23 gauge needle three times. Spin at 11, 200 g for eight minutes to clarify lysate. After centrifugation move the tubes on ice, and transfer supernatant to the new tube.

Take an aliquot, dilute it, and measure absorbance at 260 nanometer using a nano drop. If there are multiple samples, adjust the lysates to the same optical density and keep the samples on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from mouse testis.

Dissect the mouse testis. Make a small incision in the tunica albugenia and collect the seminiforous tubules of the testis and transfer them in the conical tube containing five milliliters of pbs. Mix the tissue vigorously.

And allow the tissue to settle on ice for five minutes. Remove the buffer, and wash it with pbs two more times. Transfer the seminiferous tubules in a two milliliter tube and spin them down at 500 g for one minute.

Remove the supernatant. Add 500 microliter lysis buffer to the tubules and use a pipet to disrupt the tissue. Transfer the suspension to a small volume dounce homogenizer.

Disrupt the tissue with seven to eight strokes of the glass pestle. Transfer the lysate to a micro centrifuge tube. Centrifuge the sample at 12, 000 g at four degrees Celsius for eight minutes to clear the lysate.

Transfer the supernatant into a new tube, and keep it on ice. Then, proceed with sucrose gradient preparation. Sucrose gradient preparation.

Place an ultracentrifuge tube into the microblock and draw the line along the upper level of the block. Then transfer the tube into a stable rack. Take a ten milliliter syringe, with the layering device attached and fill the syringe with 10%sucrose solution.

Gently release it at the bottom of the ultracentrifuge tube until sucrose solution reaches the mark. 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.

Then seal the tube with provided cap. To prepare sucrose gradient, turn the gradient maker device on. Then level the plate using the up or down buttons and press done.

After leveling the plate press grad. Go to the list of gradient menu and select the SW41 rotor. Then choose desired sucrose gradient.

Press use. Place magnet base tube holder on the gradient maker. Then transfer the tube into the holder.

Up to six gradients can be prepared at the same time. Press run. The gradient maker rotates the tubes at the program speed and angles, forming a linear gradient.

It will take only a few minutes to prepare the gradient. When the process is completed, place the tube in a rack. Remove the volume, equal of sample volume from the top of the ultracentrifuge tubes with sucrose gradients.

Carefully load 500 microliters of the lysate on top. Place the tubes in rotor buckets and balance them. Centrifuge at 260, 000 g for two hours at four degrees Celsius.

Polysome fractionation and sample collection. After ultracentrifugation, place the tubes in the rotor buckets on ice. Turn the fraction collector and the fractionator on.

Click scan on the fractionator menu. Put a rack with collection tubes into fraction collector. Fill up a rinse reservoir, on the side of the fractionator with deionized water.

Press rinse key for ten seconds to rinse the pump. Attach a rinse adapter with the syringe filled with water to the piston for the calibration. Open the fractionator software on the computer.

Press calibrate. Use the default setting and press Ok.Be ready to inject water from the syringe. Press Ok to do calibration.

Immediately start injecting water for the next five seconds. The sign Zero calibration completed will appear. Remove a rinse adapter with the syringe and attach a tip to the piston of the fractionator.

Open the brass air valve and press air key for ten seconds to dry air tubing and flow cell. Close the air valve. Take 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. Place the holder under the piston. Often, polysomal bands can be seen by eye.

Introduce the desired settings for fraction numbers and volume. Name the file and press Ok, then go to graph button. In the next window press start scan.

Settings will appear, and press Ok.The collector will move from the gutter to the first fraction, and the piston will move into the tube. When the piston will reach the top of the gradient it will slow to your selected speed, and fractions will be collected. Usually, we collect 24 fractions at 500 microliters each.

When completed, the piston will move out of the gradient tube. Open the brass air valve. Press air key on the fractionator to retrieve the last fraction.

At the end of the run you will see the absorbance profile of the gradient. Analysis of the fractions. Fractions obtained can be used for RNA and Protein analysis.

RNA can be analyzed by electrophoresis followed by Northern Blot or used for cDNA production, followed by RT-qPCR reaction to analyze association of individual mRNA's with polysomes. Next generation sequencing can be used to analyze the translational status of mRNA's on a transcriptome scale. For protein analysis of polysomal fractions, proteins are precipitated with tricluricedic acid to concentrate them.

Then proteins are analyzed by Western Blotting, or by Mass Spec at the proteome level.Results. Here we present the results of polysome fractionation and analysis on the examples of three organisms. Leishmania, cultured human cells, and mouse tissue.

Polysome fractionation was used to study Sherp and Tubulin mRNA's association with ribosomes during translation and Leishmania major. The absorbance graph for the fractionation has a distinct shape with typical peaks for ribosome sub-units 40S and 60S, monosomes 80S, and polysomes. Aliquots of the fractions were combined into three groups.

Pre-polysomes, light polysomes, and heavy polysomes. The mRNA relative levels were studied by a real time quantitative PCR. The data suggests that the Tubulin mRNA is actively translated.

As it was mostly associated with heavy and light polysomes. While the translation of Sherp mRNA is less active. It was mostly found in association with pre-polysomes and light polysomes.

Thus, this experiment clearly demonstrates the ability of the technique to study an individual mRNA engagement in translation. This figure presents a typical absorbance graph of the HeLa cells polysome fractionation. The proteins were concentrated by precipitation with 10%trichloroacetic acid.

The small sub unit ribosomal protein RPS6, and large sub unit ribosomal protein RPL11 were detected by Western Blot. Their distribution correlated well with the distinct peaks, on the absorbance spectrum. On this figure, we present an example of the detection of ribosomal RNAs by electrophoresis in fractionated polysomes form the mouse testis.Conclusions.

Polysomal profiling is a versatile technique, that can be used to analyze translational state of individual mRNAs, examine ribosome-associated proteins, and study translational regulation in different model organisms under different conditions.