Name: analysis of translation in the developing mouse brain using polysome profiling
Uploaded: 2021-05-22T14:00:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Analysis of Translation in the Developing Mouse Brain using Polysome Profiling at JoVE.com

This work was funded by a NSERC Discovery Grant (RGPIN/04246-2018 to G.Y.). G.Y. is a Canada Research Chair. S.K. was funded by Mitacs Globalink Graduate Fellowship and ACHRI Graduate Student Scholarship.

All animal use was supervised by the Animal Care Committee at the University of Calgary. CD1 mice used for the experiment were purchased from commercial vendor.
1. Preparation of solutions
NOTE To prevent RNA degradation, spray workbench and all equipment with RNase decontamination solution. RNase-free tips are used for the experiment. All solutions are prepared in RNase-free water.
<ol>
	<li>Prepare cycloheximide stock solution (100 mg/mL) in DMSO and store at -20 &#176;C.</li>
	<li>Prepare 2.2 M sucrose stock solution by adding 75.3 g of sucrose to RNase-free water and topping up the volume to 100 mL (for ~16 gradient preparation). The solution can be kept at -20 &#176;C for long-term storage.</li>
	<li>Prepare 10x salt solution (1 M NaCl; 200 mM Tris-HCl, pH 7.5; 50 mM MgCl2).</li>
	<li>Prepare 60% (w/v) sucrose chase solution, containing 30 g of sucrose, 5 mL of 10x salt solution and top the volume up to 50 mL with RNase-free water.</li>
	<li>Optionally, add a speck of bromophenol blue or approximately 5 &#181;L of 1% bromophenol blue in RNase-free water to the chase solution. 1.6. Store solutions at 4 &#176;C.</li>
</ol>
2. Preparation of sucrose gradient
NOTE: Accuracy in preparation of sucrose gradients is critical in obtaining consistent and reproducible results.
<ol>
	<li>To prepare six 10-50% sucrose gradients, dilute 2.2 M sucrose solution as in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose solution</td>
			<td>10%</td>
			<td>20%</td>
			<td>30%</td>
			<td>40%</td>
			<td>50%</td>
		</tr>
		<tr>
			<td>2.2 M sucrose</td>
			<td>2 mL</td>
			<td>4 mL</td>
			<td>6 mL</td>
			<td>8 mL</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>10X salt solution</td>
			<td>1.5 mL</td>
			<td>1.5 mL</td>
			<td>1.5 mL</td>
			<td>1.5 mL</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>15 &#181;L</td>
			<td>15 &#181;L</td>
			<td>15 &#181;L</td>
			<td>15 &#181;L</td>
			<td>15 &#181;L</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>11.5 mL</td>
			<td>9.5 mL</td>
			<td>7.5 mL</td>
			<td>5.5 mL</td>
			<td>3.5 mL</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>15 mL</td>
			<td>15 mL</td>
			<td>15 mL</td>
			<td>15 mL</td>
			<td>15 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Sucrose dilutions for preparations of sucrose gradients.
NOTE: Always prepare sucrose gradients in multiples of two to balance weight during ultracentrifugation.
<ol start="2">
	<li>Wipe the metal blunt end needle with RNase decontamination solution. Briefly rinse the ultracentrifuge tubes, tubing and the syringe using RNase-free water. Air-dry the tubes such that no water drop remains in the tubes as any remaining water will alter the sucrose concentration.</li>
	<li>Place the metal needle on the clamp holder and the centrifuge tube on the motorized stage. To prepare gradients consistently, a home-assembled system was used that contains a motorized stage to hold the ultracentrifuge tube and a syringe pump to inject sucrose solutions (Figure 1, see step 9 for details).</li>
	<li>Fill a 30 mL syringe with ~16 mL of 10% sucrose (enough for six gradients), place it on the syringe pump and connect it to the needle. Make sure that there are no air bubbles in the syringe, tubing, and needle. Wipe the tip off the needle to remove any residual solution.</li>
	<li>Move the motorized stage up such that the tip of the needle touches the centre of the tube at the bottom.</li>
	<li>Set the syringe pump at a flow rate of 2 mL/min for a volume of 2.3 mL.</li>
	<li>After dispensing 2.3 mL of 10% sucrose solution, move down the motorized stage and repeat the process for all six gradients.</li>
	<li>Repeat steps 2.4-2.7 to add 20% sucrose solution to the bottom of the tubes, followed by 30%, 40% and 50% sucrose solutions similarly.</li>
	<li>After preparation of the gradient, seal the ultracentrifuge tube using a paraffin film.</li>
	<li>Leave the tubes overnight at 4 &#176;C such that the different sucrose layers diffuse together to give a continuous gradient.</li>
</ol>
3. Tissue dissection
NOTE: Pregnant mice were euthanized by cervical dislocation preceded by anesthesia with 5% isoflurane.
<ol>
	<li>Collect CD1 mouse embryos at embryonic day 12, or other timepoints as needed, and place embryos in a &#216; 10 cm plate containing ice-cold Hank&#39;s Balanced Salt Solution (HBSS) on ice to retain cell viability.</li>
	<li>Under the dissection scope, transfer one embryo to a &#216; 6 cm plate containing ice-cold HBSS.</li>
	<li>Use 21-23 G needles to fix the position of the head by penetrating through eyes at an approximately 45&#176; angle and apply force to make sure the needles are fixed on the plate (Figure 2A).</li>
	<li>Use No.5 forceps to remove skin and skull, from the middle to the sides.</li>
	<li>Use forceps to cut the olfactory bulbs and remove the meninges to expose the cortical tissues.3.6.Use curved forceps to cut the cortical tissues into 2-3 mL of neurobasal medium on ice (Figure 2B). Pool cortical tissues from different embryos as needed. Tissues from 8-10 embryos usually give ~200 &#181;g total RNA.</li>
</ol>
4. Cell lysis
<ol>
	<li>On the day of tissue dissection, prepare fresh cell lysis buffer as described in Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Final concentration</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 7.5)</td>
			<td>20 mM</td>
			<td>100 &#181;L</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>100 mM</td>
			<td>250 &#181;L</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>5 mM</td>
			<td>25 &#181;L</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>1% (v/v)</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>0.5% (w/v)</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>1 mM</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>100 &#181;g/mL</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td></td>
			<td>Top up to 5 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>5 mL</td>
			<td>5 mL</td>
		</tr>
	</tbody>
</table>
Table 2: Preparation of&#160;polysome lysis buffer.
NOTE: Supplement lysis buffer with protease and phosphatase inhibitors.
<ol start="2">
	<li>Add cycloheximide to the neurobasal medium with dissected tissues to a final concentration of 100 &#181;g/mL and incubate at 37 &#176;C for 10 min. Cycloheximide blocks translation elongation and, therefore, prevents ribosome run-off 15.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Wash the tissues twice with ice-cold phosphate-buffered saline (PBS) supplemented with 100 &#181;g/mL cycloheximide.</li>
	<li>Add 500 &#181;L of cell lysis buffer supplemented with 4 &#181;L of RNase inhibitor. Pipette up and down to resuspend the tissue in lysis buffer. Use an insulin needle to gently lyse the tissue.</li>
	<li>Incubate the tissues on ice for 10 min with brief vortexing every 2-3 min. 
	NOTE: To prevent tissue degradation, ensure that all steps of tissue lysis are carried out on ice.</li>
	<li>Centrifuge at 2,000 x g for 5 min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new centrifuge tube on ice.</li>
	<li>Centrifuge at ~13,000 x g for 5 min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new centrifuge tube on ice.</li>
	<li>Measure the RNA concentration in the tissue lysate using a UV-Vis spectrophotometer. 
	NOTE: If multiple samples are included in the experiment, dilute samples to the same concentration with extra cell lysis buffer to minimize variation.</li>
</ol>
5. Sample loading and ultracentrifugation
<ol>
	<li>Pre-cool the ultracentrifugation rotor and swing buckets at 4 &#176;C, and set the temperature of the ultracentrifuge to 4 &#176;C.</li>
	<li>Keep 20 &#181;L of the tissue lysate as total RNA input.</li>
	<li>Load samples (50-300 &#181;g RNA with an equal volume) on the top of the sucrose gradients by slowly dispensing the lysate to the walls of the ultracentrifuge tubes.</li>
	<li>Gently place the ultracentrifuge tubes in the swing bucket. Ensure that all diametrically opposite buckets are balanced.</li>
	<li>Load the swing buckets on the rotor. Set the ultracentrifuge to 190,000 x g (~39,000 rpm) at 4 &#176;C for 90 min.</li>
	<li>Gently place the gradients on ice after centrifugation.</li>
</ol>
6. Fractionation and sample collection
NOTE: A home-assembled fractionating, recording and collecting system is used for the analysis and collecting samples from the gradients (Figure 3, see Device components).
<ol>
	<li>Place an empty ultracentrifuge tube on the tube piercer and gently penetrate the tube with the needle from the bottom.</li>
	<li>Switch on the UV monitor, and open the digital signal recording software.</li>
	<li>Fill a 30 mL syringe with 25 mL of 60% sucrose chase solution. Gently press the syringe such that the chase solution fills up the empty tube and go through the 254 nm UV monitor to set the baseline for detection.</li>
	<li>Press auto-zero on the UV-monitor to register a baseline for detection. Press play on the software to begin recording.</li>
	<li>Once the system establishes a baseline, pause recording on the software and retract the chase solution. Ensure that no residual chase solution remains in the system. 
	NOTE: Rinse the system with RNase-free water to remove residual sucrose solutions remaining from the previous run.</li>
	<li>Load one sample tube to the tube piercer. Gently penetrate the tube with the needle from the bottom.</li>
	<li>Begin recording on the software. Start the syringe pump (flow rate of 1 mL/min for a volume of 25 mL) and the fraction collector to collect polysome fractions. Set fractionator settings to 30 s. 
	NOTE: Before each run, ensure no air bubbles in the syringe or the tubing by gently pressing the syringe to let the chase solution flow continuously through the needle.</li>
	<li>Collect the fractions into 1.5 mL tubes (500 &#181;L each) using a fraction collector.</li>
	<li>The fractions can be processed immediately or stored at -80 &#176;C.</li>
	<li>After fraction collection, rinse the system with RNase-free water to remove any remnant sucrose.</li>
</ol>
7. Extraction of RNA
<ol>
	<li>To each fraction, add 10 ng of luciferase mRNA spike-in control.</li>
	<li>Add three volumes of guanidium hydrocholride based commercial RNA isolation reagent to each fraction.</li>
	<li>Vortex briefly for 15 s.</li>
	<li>Extract RNA using an RNA extraction kit compatible with the solution used in 7.2.</li>
</ol>
8. Reverse transcription and real-time PCR
<ol>
	<li>Measure the concentration of the RNA using UV-Vis spectrophotometer.</li>
	<li>Subject RNA to reverse transcription using a cDNA synthesis kit, according to manufacturer&#39;s protocol.</li>
	<li>Use quantitative real-time PCR (qPCR) to examine the polysomal distribution of gapdh mRNA as an example. qPCR was performed using a qPCR detection system and a qPCR mastermix reagent, with the following cycling conditions: 95 &#176;C for 10 min, then 40 cycles of 95 &#176;C for 15 s, 60 &#176;C for 30 s, 72 &#176;C for 40 s, followed by 95 &#176;C for 60 s.</li>
	<li>Obtain Threshold cycle (Ct) values from the amplification plots and used to calculate fold change using &#916;&#916;Ct method.</li>
</ol>
9. Sucrose gradient making system assembly
NOTE: Follow the steps to assemble each component (as described in Table 3) of the sucrose gradient maker (Figure 1).
<ol>
	<li>Mount two vertical brackets (A2) on the base breadboard (A1).</li>
	<li>Use two slim right-angle brackets (B2) to mount the linear stage actuator to the base breadboard (A1).</li>
	<li>Use the setscrew on the &#216;12.7 mm aluminum post (C2) to fix it on the tube holder base breadboard (C1) as a stand for the tube holder.</li>
	<li>Assemble the right-angle &#216;1/2&#34; to &#216;6 mm post clamp (C3) with the post (C2) and the mini-series optical post (C4).</li>
	<li>Use the setscrew on the optical post (C4) to connect a small V-clamp (C5) as the tube holder.</li>
	<li>Put a centrifuge tube in the tube holder and adjust the angle to make it vertical. Mark the position of the tube and mount the pedestal post holder (C6) on the base breadboard (C1) to support the tube.</li>
	<li>On the other side of the breadboard (A1), use the setscrew of the &#216;12.7 mm aluminum post (D1) to fix it and connect a mini-series optical post (D3) using a right-angle &#216;1/2&#34; to &#216;6 mm post clamp (D2).</li>
	<li>Connect the miniature V-clamp (D4) with blunt-end needle (D5) to the post (D3). Adjust the angle of the clamp to set the needle vertical, and adjust the length of post to ensure the needle meets the centrifuge tube.</li>
	<li>Connect the actuator (B1) to the stepper motor driver (E1) and use the UNO R3 controller board and the joystick module from the UNO starter kit (E2) to control the actuator. A power adaptor (e.g., 9-24 V AC/DC adjustable power adaptor) can be used to drive the motor separately.</li>
	<li>Place the syringe pump (F) next to the gradient making station and connect the syringe with the needle.</li>
	<li>Control the tube holder stage up and down using the joystick.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Item</td>
		</tr>
		<tr>
			<td>B1</td>
			<td>Linear stage actuator</td>
		</tr>
		<tr>
			<td>E1</td>
			<td>Stepper motor driver</td>
		</tr>
		<tr>
			<td>E2</td>
			<td>UNO project super starter kit</td>
		</tr>
		<tr>
			<td>A1</td>
			<td>Breadboard</td>
		</tr>
		<tr>
			<td>A2</td>
			<td>Vertical bracket</td>
		</tr>
		<tr>
			<td>B2</td>
			<td>Slim right-angle bracket</td>
		</tr>
		<tr>
			<td>C1</td>
			<td>Mini-series breadboard</td>
		</tr>
		<tr>
			<td>C5</td>
			<td>Small V-clamp</td>
		</tr>
		<tr>
			<td>D4</td>
			<td>Miniature V-clamp</td>
		</tr>
		<tr>
			<td>C2</td>
			<td>&#216;12.7 mm aluminum post</td>
		</tr>
		<tr>
			<td>C4, D3</td>
			<td>Mini-series optical post</td>
		</tr>
		<tr>
			<td>D1</td>
			<td>&#216;12.7 mm aluminum post</td>
		</tr>
		<tr>
			<td>C3, D2</td>
			<td>Right-angle &#216;1/2&#34; to &#216;6 mm post clamp</td>
		</tr>
		<tr>
			<td>C6</td>
			<td>Mini-series pedestal post holder base</td>
		</tr>
		<tr>
			<td>D5</td>
			<td>Blunt end needle</td>
		</tr>
		<tr>
			<td>F</td>
			<td>Syringe pump</td>
		</tr>
	</tbody>
</table>
Table 3: Gradient making system components.
10. Fractionating and detecting system assembly (Figure 2).
<ol>
	<li>Use a regular round jaw burette clamp to mount the optics module of the UV monitor on the tube piercer. Attach one end of the tubing (1 cm long and 0.56 mm internal diameter) to the connecter of the tube piercer and the other end to the Fluid In port of the optics module. Connect the Fluid Out port to the fractionator.</li>
	<li>Connect the optics module with the control unit of the UV monitor according the manual of the manufacturer.</li>
	<li>Use a breakout cable to connect the signal output socket to the digital converter at the ground and analog input connections, according to the manufacturer&#39;s manual.</li>
	<li>Connect the digital converter to a laptop using a regular USB cable, and record the converted digital signals using data acquisition software.</li>
</ol>

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The authors declare no competing interests.

Polysome profiling is a commonly used and powerful technique to assess the translational status at both single gene and genome-wide levels14 . In this report, we present a protocol of polysome profiling using a home-assembled platform and its application to analyze the developing mouse cortex. This cost-effective platform is easy to assemble and generate robust, reproducible sucrose gradients and polysome profiling with high sensitivity.
It is worthy to note that the preparation of consistent and good quality sucrose gradients is critical to obtain reproducible polysome profiling results17. Changes in the volume of 10-50% sucrose solutions during gradient preparation could cause the shift of the polysome peaks and inconsistency of results in downstream analysis. Moreover, disturbing the gradient during the insertion and removal of the needle could contribute to the inconsistency of gradient preparation. Compared to other approaches and commercial devices, our home-made gradient making system used a motorized stage to reduce disturbance of gradients during preparation and a syringe pump to accurately dispense sucrose solutions, which offers a cheap and simple solution for gradient preparation.
Applying the polysome profiling platform and protocol present here to the developing mouse cortex, we found that the ribosomal protein Rpl10 and cytoplasmic proteins Gapdh and Csde1 were distributed in the correct fractions. Moreover, the highly translated gapdh and sox2 mRNAs were enriched in polysomal fractions, while translationally repressed rpl34 and rpl7 mRNAs showed less enrichement in polysomes, which validate our platform and protocol. With a similar approach, the translational status of other genes in the developing cortex can be determined individually by real-time PCR. Of note, polysome profiling has been used to analyse translational status in the developing brain at the genome-wide level. In this regard, fractions containing polysomes can be combined and extracted RNA can be analysed using deep sequencing. The platform and protocol present here can be adapted and further optimized to suit translatomic studies using other cell types and tissues. Considering the availability of cortical tissues limited by experimental conditions (e.g., &#60;50 &#181;g RNA), additional optimization of the protocol may provide further increases in the efficiency and reproducibility of the experiments.
While polysome profiling provides insights into the translational status of mRNAs in the developing cortex, it has limitations to analyze specific cell types at later developmental stages, when different neural cell types are present. To address this question, polysome profiling can be integrated with TRAP by pull-down of epitope tagged ribosomal protein expressed under a tissue specific promoter11.
In conclusion, the platform and protocol that we report here for sucrose gradient making and fractionation provide an economical solution to polysome profiling experiments in the context of brain development as well as other biological contexts.

During the development of the mammalian brain, neural stem cells proliferate and differentiate to generate neurons and glia1,2 . The perturbation of this process can lead to alterations in brain structure and function, as seen in many neurodevelopmental disorders3,4. The proper behavior of neural stem cells requires the orchestrated expression of specific genes5. While the epigenetic and transcriptional control of these genes has been intensively studied, recent findings suggest that gene regulation at other levels also contributes to the coordination of neural stem cell proliferation and differentiation6,7,8,9,10. Thus, addressing the translational control programs will greatly advance our understanding of the mechanisms underlying neural stem cell fate decision and brain development.
Three main techniques with different strengths have been widely applied to assess the translational status of mRNA, including ribosome profiling, translating ribosome affinity purification (TRAP) and polysome profiling. Ribosome profiling uses RNA sequencing to determine ribosome-protected mRNA fragments, allowing the global analysis of the number and location of translating ribosomes on each transcript to indirectly infer the translation rate by comparing it to transcript abundance11. TRAP takes advantage of epitope-tagged ribosomal proteins to capture ribosome-bound mRNAs12. Given that the tagged ribosomal proteins can be expressed in specific cell types using genetic approaches, TRAP allows the analysis of translation in a cell type-specific manner. In comparison, polysome profiling, which uses sucrose density gradient fractionation to separate free and poorly-translated portion (lighter monosomes) from those being actively translated by ribosomes (heavier polysomes), provides a direct measurement of ribosome density on mRNA13. One advantage this technique offers is its versatility to study the translation of specific mRNA of interest as well as genome-wide translatome analysis14.
In this paper, we describe a detailed protocol of polysome profiling to analyze the developing mouse cerebral cortex. We use a home-assembled system to prepare sucrose density gradients and collect fractions for downstream applications. The protocol presented here can be adapted easily to analyze other types of tissues and organisms.

<table><tbody><tr><td>1.5 mL RNA free microtubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>10 cm dish</td><td>Greiner-Bio</td><td>664160</td><td></td></tr><tr><td>1M MgCl2</td><td>Invitrogen</td><td>AM9530G</td><td></td></tr><tr><td>21-23G needle</td><td>BD</td><td>305193</td><td></td></tr><tr><td>2M KCl</td><td>Invitrogen</td><td>AM8640G</td><td></td></tr><tr><td>30 mL syringe</td><td>BD</td><td>302832</td><td></td></tr><tr><td>Blunt end needle</td><td>VWR</td><td>20068-781</td><td></td></tr><tr><td>Breadboard</td><td>Thorlabs</td><td>MB2530/M</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>115-39-9</td><td></td></tr><tr><td>CD1 mouse</td><td></td><td>Charles River Laboratory</td><td></td></tr><tr><td>Curved tip forceps</td><td>Sigma</td><td>#Z168785</td><td></td></tr><tr><td>Cycloheximide</td><td></td><td>Sigma</td><td>66-81-9</td></tr><tr><td>Data acquisition software TracerDAQ</td><td>Measurement Computing</td><td></td><td></td></tr><tr><td>Digital converter</td><td></td><td>Measurement Computing</td><td>USB-1208LS</td></tr><tr><td>Direct-zol RNA miniprep kit</td><td>Zymo</td><td>R2070</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-basic</td><td>12-03-3483</td><td></td></tr><tr><td>DMSO</td><td>Bioshop</td><td>67-68-5</td><td></td></tr><tr><td>Dumont No.5 forceps</td><td>Sigma</td><td>#F6521</td><td></td></tr><tr><td>Fraction collector</td><td>Bio-Rad</td><td>Model 2110</td><td></td></tr><tr><td>HBSS</td><td>Wisent</td><td>311-513-CL</td><td></td></tr><tr><td>Linear stage actuator</td><td>Rattmmotor</td><td>CBX1605-100A</td><td></td></tr><tr><td>Luciferase control RNA</td><td>Promega</td><td>L4561</td><td></td></tr><tr><td>Maxima first strand cDNA synthesis kit</td><td>Themo Fisher</td><td>M1681</td><td></td></tr><tr><td>Miniature V-clamp</td><td>Thorlabs</td><td>VH1/M</td><td></td></tr><tr><td>Mini-series breadboard</td><td>Thorlabs</td><td>MSB7515/M</td><td></td></tr><tr><td>Mini-series optical post</td><td>Thorlabs</td><td>MS2R/M</td><td></td></tr><tr><td>Mini-series pedestal post holder base</td><td>Thorlabs</td><td>MBA1</td><td></td></tr><tr><td>NaCl</td><td>Bio-basic</td><td>7647-14-5</td><td></td></tr><tr><td>Neurobasal media</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>&amp;Oslash;12.7 mm aluminum post</td><td>Thorlabs</td><td>TRA150/M</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM992</td><td></td></tr><tr><td>PerfeCTa SYBR green fastmix</td><td>Quanta Bio</td><td>CA101414-274</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Wisent</td><td>311-010-CL</td><td></td></tr><tr><td>Puromycin</td><td>Bioshop</td><td>58-58-2</td><td></td></tr><tr><td>Right-angle clamp</td><td>Thorlabs</td><td>RA90/M</td><td></td></tr><tr><td>Right-angle &amp;Oslash;1/2&quot; to &amp;Oslash;6 mm post clamp</td><td>Thorlabs</td><td>RA90TR/M</td><td></td></tr><tr><td>Rnase AWAY</td><td>Molecular BioProducts</td><td>7002</td><td></td></tr><tr><td>RNase free tips</td><td>Frogga Bio</td><td>FT10, FT200, FT1000</td><td></td></tr><tr><td>RNase free water</td><td>Wisent</td><td>809-115-CL</td><td></td></tr><tr><td>RNasin</td><td>Promega</td><td>N2111</td><td></td></tr><tr><td>Slim right-angle bracket</td><td>Thorlabs</td><td>AB90B/M</td><td></td></tr><tr><td>Small V-clamp</td><td>Thorlabs</td><td>VC1/M</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>302-95-4</td><td></td></tr><tr><td>Stepper motor driver</td><td>SongHe</td><td>TB6600</td><td></td></tr><tr><td>Sucrose</td><td>Bioshop</td><td>57501</td><td></td></tr><tr><td>SW 41 Ti rotor</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-4500</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-4500</td><td></td></tr><tr><td>Triton-X-100</td><td>Bio-basic</td><td>9002-93-1</td><td></td></tr><tr><td>Trizol</td><td>Thermofisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Tube piercer</td><td>Brandel</td><td>BR-184</td><td></td></tr><tr><td>Ultracentrifuge</td><td></td><td>Beckman Coulter</td><td>L8-70M</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>UltraPure 1M Tris-HCl pH 7.5</td><td>Invitrogen</td><td>15567-027</td><td></td></tr><tr><td>UNO project super starter kit</td><td>Elegoo</td><td>EL-KIT-003</td><td></td></tr><tr><td>UV monitor</td><td>Bio-Rad</td><td>EM-1 Econo</td><td></td></tr><tr><td>Vertical bracket</td><td>Thorlabs</td><td>VB01A/M</td><td></td></tr></tbody></table>

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.

As a demonstration, the cortical lysate containing 75 &#181;g RNA (pooled from 8 embryos) was separated by the sucrose gradient into 12 fractions. Peaks of UV absorbance at 254 nm identified fractions containing the 40S subunit, 60S subunit, 80S monosome and polysomes (Figure 4A). Analysis of fractions by western blot for the large ribosomal subunit, Rpl10 showed its presence in the 60S subunit (fraction 3), monosome (fraction 4) and polysomes (fractions 5-12) (Figure 4B). In contrast, cytoplasmic proteins Gapdh and Csde1 were not associated with ribosomes but were enriched in fractions containing free RNA (fraction 1) (Figure 4B). Consistent with the separation of proteins in different fractions, we found that gapdh and sox2 mRNAs were highly enriched in the fractions containing heavy polysomes (more than three ribosomes in fractions 7-12), suggesting that gapdh and sox2 mRNAs are efficiently translated in the developing cortex (Figure 4C,D). In contrast, rpl7 and rpl34 mRNAs were enriched in the fraction containing monosomes, suggesting repressed translation (Figure 4E,F)16. These results validated our polysome fractionation protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62088/62088fig01.jpg" /> 
Figure 1: Setup for sucrose gradient preparation. (A)&#160;Home-assembled sucrose gradient maker consisting of a linear stage actuator, a tube holder on the motorized stage, a stage controller set, a metal blunt-end needle mounted on a needle holder, and an automated syringe pump with a syringe connected to the needle (see Table 3). (B) To prepare the sucrose gradient, the ultracentrifuge tube is placed in the tube holder. Sucrose solutions are dispensed through the blunt-end needle using a syringe pump. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62088/62088fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62088/62088fig02.jpg" /> 
Figure 2: Dissection of the cortical tissues from the development mouse embryo. (A) Image showing an E12.5 CD1 embryo fixed to a &#216; 6 cm plate using 21G-23G needles. (B)&#160;Image showing the dissected cortex after removal of the skin, skull and meninges (marked with white dashed lines). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62088/62088fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62088/62088fig03.jpg" /> 
Figure 3: Setup for fractionating, recording and sample collecting. Image depicting the home-assembled fractionation, recording and sample collection system comprising of an automated syringe pump, a tube piercer, a fraction collector and a UV monitor with a digital convertor connected to a laptop for the continuous monitoring of UV absorbance. Data acquisition is handled by the commercial data acquisition software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62088/62088fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62088/62088fig04.jpg" /> 
Figure 4: Polysome profiling analysis of the developing mouse cortex. (A)&#160;UV absorbance showing fractions containing free RNA (fraction 1), 40S subunit (fraction 2), 60S subunit (fraction 3), 80S monosomes (fraction 4) and polysomes (fraction 5-12). 75 &#181;g total RNA pooled from 8 embryos was used. (B)&#160;Western blots showing the distributions of Gapdh, Rpl10 and Csde1 proteins in fractions. qPCR analysis showing the distribution of gapdh (C), sox2 (D), rpl7 (E)&#160;and rpl34 (F) mRNAs in fractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62088/62088fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Translation in the Developing Mouse Brain using Polysome Profiling at JoVE.com

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

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 ...

analysis-translation-developing-mouse-brain-using-polysome

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.0K Views.  University of Calgary. Translationele regulatie speelt een belangrijke rol in de ontwikkeling van de hersenen. Polysomale profilering biedt een nuttig en krachtig hulpmiddel om de translationele status van mRNA te beoordelen, waardoor het mogelijk is om translationele controlefuncties tijdens de hersenontwikkeling te onderzoeken. Deze methode is een eenvoudige en goedkope aanpak om sucrosegradiënten te maken en polysoomfractie te beoordelen.In dit protocol wordt het gebruikt om de zich ontwikkelende muissch...

Video: Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Biochemistry

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

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

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

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

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

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

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

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. ...

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. ...

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 ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

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